Actriib proteins and variants and uses therefore relating to utrophin induction for muscular dystrophy therapy

ABSTRACT

In certain aspects, the present invention provides compositions and methods for inducing utrophin expression in muscle with an ActRIIB protein as therapy for muscular dystrophy. The present invention also provides methods of screening compounds that modulate activity of an ActRIIB protein and/or an ActRIIB ligand.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/459,338, filed Mar. 15, 2017 (now allowed), which is a continuationof U.S. application Ser. No. 14/254,560, filed Apr. 16, 2014 (now U.S.Pat. No. 9,617,319), which is a continuation of U.S. application Ser.No. 12/948,411, filed Nov. 17, 2010 (now U.S. Pat. No. 8,710,016), whichclaims the benefit of Provisional Application Nos. 61/331,686, filed onMay 5, 2010 (now expired), 61/318,126, filed on Mar. 26, 2010 (nowexpired), and 61/281,386, filed Nov. 17, 2009 (now expired). Thespecifications of each of the foregoing applications are incorporatedherein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 24, 2021, isnamed 1848179-057-104_Seq.txt and is ?????? bytes in size.

BACKGROUND OF THE INVENTION

Duchenne's Muscular Dystrophy (DMD) is a genetic disease that resultsfrom a variety of different mutations in the gene coding for dystrophin.Dystrophin is a large cytoskeletal protein that provides structuralintegrity to contracting muscle fibers. DMD patients produce little, ifany, functional dystrophin, resulting in fragility of the sarcolemmalmembranes that surround each muscle fiber. As a consequence of thisfragility, DMD patients exhibit progressive degeneration of skeletal andcardiac muscles, with onset typically at age two to six. The diseasecauses generalized weakness and muscle wasting. Survival is rare beyondthe early 30s.

In a related and somewhat milder condition, Becker muscular dystrophy(BMD), the patient produces some functional dystrophin, but not enoughto provide normal durability and maintenance of muscle tissue. BMDpatients usually have a longer lifespan than DMD patients.

While DMD and BMD are presently incurable diseases, one therapeuticapproach under investigation involves treatment with agents thatincrease the levels of a protein called utrophin. Utrophin isstructurally and functionally similar to dystrophin. Moreover, utrophinis normally present in muscle fibers during fetal development andremains in the mature fibers at neuromuscular junctions. At sufficientlevels and with appropriate localization to the sarcolemma, utrophinshows evidence of partially compensating for the absence of dystrophinin animal models of DMD.

DMD and BMD patients have a normal utrophin gene, and it may be possibleto increase the strength of muscle fibers in patients by increasingutrophin production.

Thus, there is a need for agents that increase utrophin productionand/or sarcolemmal localization of utrophin.

SUMMARY OF THE INVENTION

In certain aspects, the present disclosure provides methods forincreasing utrophin expression and/or localization at the cell membrane(sarcolemma) of muscle fibers in DMD and BMD patients by usingantagonists of the ActRIIB signaling pathway. Such antagonists may be,for example, soluble ActRIIB proteins (e.g., ActRIIB-Fc fusionproteins), antagonists that bind to ActRIIB or inhibit ActRIIBexpression, and antagonists that bind to or inhibit the expression ofligands that signal through ActRIIB and participate in the regulation ofutrophin expression in skeletal or cardiac muscle. Such ligands includemyostatin, GDF3, activins, BMP7, BMP2 and BMP4. In particular, thedisclosure demonstrates that ActRIIB-Fc fusion proteins increasesarcolemmal expression of utrophin in a mouse model of musculardystrophy. By increasing the resistance of the sarcolemma to damage,antagonists of the ActRIIB signaling pathway may decrease the cycle ofmuscle injury, inflammation and degradation that is the hallmark ofdystrophin-deficient conditions, such as DMD and BMD. These beneficialeffects are combined with the pronounced effects of ActRIIB antagonistson overall muscle mass and strength. As a consequence, the disclosureprovides a paradigm shift for the use of ActRIIB pathway antagonists inthe management of muscle disorders, moving from a paradigm emphasizingthe increase of muscle fiber size and strength to a paradigm thatacknowledges an increase in muscle fiber integrity, a feature that isparticularly relevant in muscular dystrophies. A consequence of musclefragility in muscular dystrophy patients is an increase in serum markersof muscle degeneration such as creatine kinase (particularly isoform MM,also referred to as CK-MM). The data provided herein indicate thatActRIIB pathway antagonists can increase muscle fiber integrity andtherefore decrease the level of serum markers, such as CK-MM. Therefore,markers of muscle degeneration, such as serum CK-MM levels, may be usedas a mechanism for monitoring efficacy of such therapies in DMD and BMDpatients. For example, failure to decrease a marker of muscledegeneration may be used as an indicator to increase dose or terminatedosing for lack of benefit, and successful decrease of a marker ofmuscle degeneration may be used as an indicator that a successful dosehas been reached. Similarly, a patient with an elevated marker of muscledegeneration may be a particularly appropriate candidate for treatmentwith ActRIIB antagonists. As described in Zatz et al. (J. Neurol. Sci.1991 102(2):190-6), CK levels reach maxima in DMD and BMD patientsduring a time period of maximum muscle degeneration (typically in an agerange of 1 to 6, 7, or 8 years of age in DMD and 10 to 15 years of agein BMD), and thus DMD and BMD patients with high levels of a marker ofmuscle degeneration (elevated even as compared to others with thedisease state, e.g., serum CK-MM levels higher than 50%, 60%, 70%, 80%,90% of other patients with such disease) are particularly appropriatepatients for treatment with ActRIIB antagonists such as ActRIIB-Fcproteins.

In certain aspects, the disclosure provides methods for increasingsarcolemmal expression of utrophin by administering to a patient in needthereof an effective amount of an ActRIIB-related polypeptide. AnActRIIB-related polypeptide may be an ActRIIB polypeptide (e.g., anActRIIB extracellular domain or portion thereof) that binds to anActRIIB ligand such as GDF3, BMP2, BMP4, BMP7, GDF8, GDF11, activin ornodal. Optionally, the ActRIIB polypeptide binds to an ActRIIB ligandwith a Kd less than 10 micromolar or less than 1 micromolar, 100, 10 or1 nanomolar. A variety of suitable ActRIIB polypeptides have beendescribed in the following published PCT patent applications, all ofwhich are incorporated by reference herein: WO 00/43781, WO 04/039948,WO 06/012627, WO 07/053775, WO 08/097541, and WO 08/109167. Optionally,the ActRIIB polypeptide inhibits ActRIIB signaling, such asintracellular signal transduction events triggered by an ActRIIB ligand.A soluble ActRIIB polypeptide for use in such a preparation may be anyof those disclosed herein, such as a polypeptide having an amino acidsequence selected from SEQ ID NOs: 1, 2, 5, 12, and 23, or having anamino acid sequence that is at least 80%, 85%, 90%, 95%, 97% or 99%identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, 5,12, and 23. A soluble ActRIIB polypeptide may include a functionalfragment of a natural ActRIIB polypeptide, such as one comprising atleast 10, 20 or 30 amino acids of a sequence selected from SEQ ID NOs:1, 2, 5, 12, and 23, or a sequence of SEQ ID NO: 1, lacking theC-terminal 1, 2, 3, 4, 5 or 10 to 15 amino acids and lacking 1, 2, 3, 4or 5 amino acids at the N-terminus. Optionally, polypeptides willcomprise a truncation relative to SEQ ID NO: 1 of between 2 and 5 aminoacids at the N-terminus and no more than 3 amino acids at theC-terminus. Another polypeptide is that presented as SEQ ID NO: 12. Asoluble ActRIIB polypeptide may include one, two, three, four, five ormore alterations in the amino acid sequence (e.g., in the ligand-bindingdomain) relative to a naturally occurring ActRIIB polypeptide. Thealteration in the amino acid sequence may, for example, alterglycosylation of the polypeptide when produced in a mammalian, insect orother eukaryotic cell or alter proteolytic cleavage of the polypeptiderelative to the naturally occurring ActRIIB polypeptide. A solubleActRIIB polypeptide may be a fusion protein that has, as one domain, anActRIIB polypeptide (e.g., a ligand-binding domain of an ActRIIB or avariant thereof) and one or more additional domains that provide adesirable property, such as improved pharmacokinetics, easierpurification, targeting to particular tissues, etc. For example, adomain of a fusion protein may enhance one or more of in vivo stability,in vivo half life, uptake/administration, tissue localization ordistribution, formation of protein complexes, multimerization of thefusion protein, and/or purification. A soluble ActRIIB fusion proteinmay include an immunoglobulin constant domain, such as an Fc domain(wild-type or mutant) or a serum albumin. In certain embodiments, anActRIIB-Fc fusion comprises a relatively unstructured linker positionedbetween the Fc domain and the extracellular ActRIIB domain. Thisunstructured linker may correspond to the roughly 15 amino acidunstructured region at the C-terminal end of the extracellular domain ofActRIIB (the “tail”), or it may be an artificial sequence of between 5and 15, 20, 30, 50 or more amino acids that are relatively free ofsecondary structure. A linker may be rich in glycine and prolineresidues and may, for example, contain repeating or non-repeatingsequences of threonine/serine and/or glycines (e.g., TG₄ (SEQ ID NO: 6),TG₃ (SEQ ID NO: 24), SG₄ (SEQ ID NO: 25), SG₃ (SEQ ID NO: 26), G₄ (SEQID NO: 27), G₃, G₂, G). A fusion protein may include a purificationsubsequence, such as an epitope tag, a FLAG tag, a polyhistidinesequence, and a GST fusion. Optionally, a soluble ActRIIB polypeptideincludes one or more modified amino acid residues selected from: aglycosylated amino acid, a PEGylated amino acid, a farnesylated aminoacid, an acetylated amino acid, a biotinylated amino acid, an amino acidconjugated to a lipid moiety, and an amino acid conjugated to an organicderivatizing agent. In general, it is preferable that an ActRIIB proteinbe expressed in a mammalian cell line that mediates suitably naturalglycosylation of the ActRIIB protein so as to diminish the likelihood ofan unfavorable immune response in a patient. Human and CHO cell lineshave been used successfully, and it is expected that other commonmammalian expression vectors will be useful.

In certain aspects, a compound disclosed herein may be formulated as apharmaceutical preparation. A pharmaceutical preparation may alsoinclude one or more additional compounds such as a compound that is usedto treat an ActRIIB-associated disorder. Preferably, a pharmaceuticalpreparation is substantially pyrogen free.

In certain aspects, the disclosure provides nucleic acids encoding asoluble ActRIIB polypeptide, which do not encode a complete ActRIIBpolypeptide. An isolated polynucleotide may comprise a coding sequencefor a soluble ActRIIB polypeptide, such as described above. For example,an isolated nucleic acid may include a sequence coding for anextracellular domain (e.g., ligand-binding domain) of an ActRIIB and asequence that would code for part or all of the transmembrane domainand/or the cytoplasmic domain of an ActRIIB, but for a stop codonpositioned within the transmembrane domain or the cytoplasmic domain, orpositioned between the extracellular domain and the transmembrane domainor cytoplasmic domain. For example, an isolated polynucleotide maycomprise a full-length ActRIIB polynucleotide sequence such as SEQ IDNO: 4 (FIG. 4), or a partially truncated version, said isolatedpolynucleotide further comprising a transcription termination codon atleast six hundred nucleotides before the 3′-terminus or otherwisepositioned such that translation of the polynucleotide gives rise to anextracellular domain optionally fused to a truncated portion of afull-length ActRIIB. Nucleic acids disclosed herein may be operablylinked to a promoter for expression, and the disclosure provides cellstransformed with such recombinant polynucleotides. Preferably the cellis a mammalian cell such as a CHO cell.

In certain aspects, the disclosure provides methods for making a solubleActRIIB polypeptide. Such a method may include expressing any of thenucleic acids (e.g., SEQ ID NO: 3) disclosed herein in a suitable cell,such as a Chinese hamster ovary (CHO) cell. Such a method may comprise:a) culturing a cell under conditions suitable for expression of thesoluble ActRIIB polypeptide, wherein said cell is transformed with asoluble ActRIIB expression construct; and b) recovering the solubleActRIIB polypeptide so expressed. Soluble ActRIIB polypeptides may berecovered as crude, partially purified or highly purified fractionsusing any of the well known techniques for obtaining protein from cellcultures.

In certain aspects, increasing sarcolemmal expression of utrophin usinga compound described herein may be useful in the treatment of musculardystrophies in which the dystrophin protein is absent, deficient, ordefective. Examples include treatment of Duchenne muscular dystrophy andBecker muscular dystrophy.

In certain aspects, the disclosure provides methods for antagonizingactivity of an ActRIIB polypeptide or an ActRIIB ligand (e.g., GDF8,GDF11, activin, BMP7, and Nodal) in a cell. The methods comprisecontacting the cell with a soluble ActRIIB polypeptide. Optionally, theactivity of the ActRIIB polypeptide or the ActRIIB ligand is monitoredby a signaling transduction mediated by the ActRIIB/ActRIIB ligandcomplex, for example, by monitoring cell proliferation, hypertrophy, orthe level of utrophin expression or localization of utrophin. The cellsof the methods include an a myocyte and a muscle cell.

In certain aspects, the disclosure provides uses of a soluble ActRIIBpolypeptide for making a medicament for the treatment of a disorder orcondition as described herein.

In certain aspects, the disclosure provides methods for increasingsarcolemmal expression of utrophin in a patient in need thereof, andsuch method may comprise administering an effective amount of a compoundselected from the group consisting of: a polypeptide comprising an aminoacid sequence that is at least 90% identical to the sequence of aminoacids 29-109 of SEQ ID NO: 2 and a polypeptide encoded by a nucleic acidthat hybridizes under stringent hybridization conditions to a nucleicacid of SEQ ID NO: 3 (FIG. 3). The polypeptide may be a fusion proteincomprising a heterologous portion. The polypeptide may be a dimer. Thepolypeptide may be fused to a constant domain of an immunoglobulin. Thepolypeptide may be fused to an Fc portion of an immunoglobulin, such asan IgG1, IgG2, IgG3 or IgG4. The polypeptide may comprise an amino acidsequence that is at least 80%, 90%, 93%, 95%, 97%, 98%, 99% or 100%identical to the sequence of amino acids 29-109, 29-128, 29-131, 29-134,25-109, 25-128, 25-131, 25-134 or 20-134 of SEQ ID NO: 2 (FIG. 2). Thepolypeptide may comprise an amino acid sequence that is at least 80%,90%, 93%, 95%, 97%, 98%, 99% or 100% identical to the sequence of aminoacids of SEQ ID NO: 5, 12, or 23. A patient to be treated with such acompound may be one having a disorder described herein, including, forexample, a muscular dystrophy.

In certain aspects, the disclosure provides methods for increasingsarcolemmal expression of utrophin in a patient in need thereof, themethod comprising administering an effective amount of a compound thatinhibits the ActRIIB signaling pathway, either by targeting ActRIIB or aligand that signals through ActRIIB. Examples of such compounds includeantagonists of ActRIIB; antagonists of myostatin; antagonists of BMP7;antagonists of BMP2; antagonists of BMP4 and antagonists of GDF3.Antagonists of each of the foregoing may be an antibody or other proteinthat specifically binds to and inhibits such target (e.g., an antibodysuch as a monoclonal antibody, or a propeptide in the case of myostatinand GDF3). Antagonists of the foregoing may also be a compound, such asa nucleic acid based compound (e.g., an antisense or RNAi nucleic acid)that inhibits the expression of ActRIIB or the ligand. A patient to betreated with such a compound may be one having a disorder describedherein, including, for example, a muscular dystrophy.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a human ActRIIB soluble (extracellular) polypeptidesequence (SEQ ID NO: 1). The C-terminal “tail” is underlined.

FIG. 2 shows human ActRIIB precursor protein sequence (SEQ ID NO: 2).The signal peptide is underlined; the extracellular domain is in bold(also referred to as SEQ ID NO: 1); and the potential N-linkedglycosylation sites are boxed.

FIG. 3 shows a nucleic acid sequence encoding a human ActRIIB soluble(extracellular) polypeptide, designated as SEQ ID NO: 3.

FIG. 4 shows a nucleic acid sequence encoding human ActRIIB precursorprotein, designated as SEQ ID NO: 4.

FIG. 5 shows an alignment of human ActRIIA (SEQ ID NO:14) and ActRIIB(SEQ ID NO: 30) with the residues that are deduced herein, based oncomposite analysis of multiple ActRIIB and ActRIIA crystal structures todirectly contact ligand (the ligand binding pocket) indicated withboxes.

FIG. 6 shows a multiple sequence alignment of various vertebrate ActRIIBproteins and human ActRIIA (SEQ ID NOs: 15-22).

FIG. 7 shows the full amino acid sequence of ActRIIB(25-131)-hFc (SEQ IDNO: 31). The TPA leader (residues 1-22) and truncated ActRIIBextracellular domain (native residues 25-131) are each underlined.Highlighted is the glutamate revealed by sequencing to be the N-terminalamino acid of the mature fusion protein.

FIG. 8 shows a nucleotide sequence encoding ActRIIB(25-131)-hFc (thecoding strand is shown at top (SEQ ID NO: 32) and the complement shownat bottom 3′-5′). Sequences encoding the TPA leader (nucleotides 1-66)and ActRIIB extracellular domain (nucleotides 73-396) are underlined.The corresponding amino acid sequence for ActRIIB(25-131) (SEQ ID NO:33) is also shown.

FIG. 9 shows the effect of ActRIIB(20-134)-mFc treatment for 20 weeks onutrophin protein levels in muscle in an mdx mouse model of musculardystrophy. A. Western blot analysis of utrophin protein in homogenatesof pectoralis major muscle. B. Densitometric quantitation of utrophinbands shown in A, normalized to GAPDH (glyceraldehyde 3-phosphatedehydrogenase) protein and expressed in relative units (RU). Data aremeans±SEM; n=5 mice per group; *, p<0.05. Chronic treatment ofmiddle-aged mdx mice with ActRIIB(20-134)-mFc increased utrophin levelsin pectoralis muscle by more than 80%.

FIG. 10 shows the effect of ActRIIB(20-134)-mFc treatment for 20 weekson utrophin distribution in muscle fibers in an mdx mouse model ofmuscular dystrophy. Shown are transverse sections through the extensordigitorum longus (EDL). Scale bar, 50 μm. ActRIIB(20-134)-mFc treatmentbroadly increased sarcolemmal levels of utrophin. A. mdx mice weretreated with vehicle. B. mdx mice were treated with ActRIIB(20-134)-mFc.

FIG. 11 shows enlargements from FIG. 10 for greater clarity. Scale bar,50 μm. A. mdx mice were treated with vehicle. B. mdx mice were treatedwith ActRIIB(20-134)-mFc.

FIG. 12 show the effect of ActRIIB(20-134)-mFc on serum creatine kinaselevels. A. shows the experimental design for the study described inExample 7. B. The graph shows serum creatine kinase levels for each ofthe four experimental groups. Group 4 had statistically lower serumcreatine kinase levels than each of Groups 1 through 3.

DETAILED DESCRIPTION 1. Overview

In certain aspects, the present invention relates to ActRIIBpolypeptides. As used herein, the term “ActRIIB” refers to a family ofactivin receptor type IIB (ActRIIB) proteins and ActRIIB-relatedproteins, derived from any species. Members of the ActRIIB family aregenerally all transmembrane proteins, composed of a ligand-bindingextracellular domain with cysteine-rich region, a transmembrane domain,and a cytoplasmic domain with predicted serine/threonine kinasespecificity. Amino acid sequences of human ActRIIA precursor protein(SEQ ID NO: 14, provided for comparison) and ActRIIB precursor proteinare illustrated in FIG. 5.

The term “ActRIIB polypeptide” is used to refer to polypeptidescomprising any naturally occurring polypeptide of an ActRIIB familymember as well as any variants thereof (including mutants, fragments,fusions, and peptidomimetic forms) that retain a useful activity. Forexample, ActRIIB polypeptides include polypeptides derived from thesequence of any known ActRIIB having a sequence at least about 80%identical to the sequence of an ActRIIB polypeptide, and preferably atleast 85%, 90%, 95%, 97%, 99% or greater identity.

In a specific embodiment, the invention relates to soluble ActRIIBpolypeptides. As described herein, the term “soluble ActRIIBpolypeptide” generally refers to polypeptides comprising anextracellular domain of an ActRIIB protein. The term “soluble ActRIIBpolypeptide,” as used herein, includes any naturally occurringextracellular domain of an ActRIIB protein as well as any variantsthereof (including mutants, fragments and peptidomimetic forms) thatretain a useful activity. For example, the extracellular domain of anActRIIB protein binds to a ligand and is generally soluble. Examples ofsoluble ActRIIB polypeptides include ActRIIB soluble polypeptidesillustrated in FIG. 1 (SEQ ID NO: 1). Other examples of soluble ActRIIBpolypeptides comprise a signal sequence in addition to the extracellulardomain of an ActRIIB protein (see Example 1). The signal sequence can bea native signal sequence of an ActRIIB, or a signal sequence fromanother protein, such as a tissue plasminogen activator (TPA) signalsequence or a honey bee melatin (HBM) signal sequence.

TGF-β signals are mediated by heteromeric complexes of type I and typeII serine/threonine kinase receptors, which phosphorylate and activatedownstream Smad proteins upon ligand stimulation (Massagué, 2000, Nat.Rev. Mol. Cell Biol. 1:169-178). These type I and type II receptors areall transmembrane proteins, composed of a ligand-binding extracellulardomain with cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine specificity. Type Ireceptors are essential for signaling; and type II receptors arerequired for binding ligands and for expression of type I receptors.Type I and II activin receptors form a stable complex after ligandbinding, resulting in phosphorylation of type I receptors by type IIreceptors.

Two related type II receptors, ActRIIA and ActRIIB, have been identifiedas the type II receptors for activins (Mathews and Vale, 1991, Cell65:973-982; Attisano et al., 1992, Cell 68: 97-108). Besides activins,ActRIIA and ActRIIB can biochemically interact with several other TGF-βfamily proteins, including BMP7, Nodal, GDF8, and GDF11 (Yamashita etal., 1995, J. Cell Biol. 130:217-226; Lee and McPherron, 2001, Proc.Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol. Cell 7:949-957; Oh et al., 2002, Genes Dev. 16:2749-54). Applicants have foundthat soluble ActRIIA-Fc fusion proteins and ActRIIB-Fc fusion proteinshave substantially different effects in vivo, with ActRIIA-Fc havingprimary effects on bone and ActRIIB-Fc having primary effects onskeletal muscle.

In certain embodiments, the present invention relates to antagonizing aligand of ActRIIB receptors (also referred to as an ActRIIB ligand) witha subject ActRIIB polypeptide (e.g., a soluble ActRIIB polypeptide).Thus, compositions and methods of the present invention are useful fortreating disorders associated with abnormal activity of one or moreligands of ActRIIB receptors. Exemplary ligands of ActRIIB receptorsinclude some TGF-β family members, such as activin, Nodal, GDF8, GDF11,and BMP7.

Activins are dimeric polypeptide growth factors and belong to theTGF-beta superfamily. There are three activins (A, B, and AB) that arehomo/heterodimers of two closely related β subunits (β_(A)β_(A),β_(B)β_(B), and β_(A)β_(B)). In the TGF-beta superfamily, activins areunique and multifunctional factors that can stimulate hormone productionin ovarian and placental cells, support neuronal cell survival,influence cell-cycle progress positively or negatively depending on celltype, and induce mesodermal differentiation at least in amphibianembryos (DePaolo et al., 1991, Proc SocEp Biol Med. 198:500-512; Dysonet al., 1997, Curr Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol.55:953-963). Moreover, erythroid differentiation factor (EDF) isolatedfrom the stimulated human monocytic leukemic cells was found to beidentical to activin A (Murata et al., 1988, PNAS, 85:2434). It wassuggested that activin A acts as a natural regulator of erythropoiesisin the bone marrow. In several tissues, activin signaling is antagonizedby its related heterodimer, inhibin. For example, during the release offollicle-stimulating hormone (FSH) from the pituitary, activin promotesFSH secretion and synthesis, while inhibin prevents FSH secretion andsynthesis. Other proteins that may regulate activin bioactivity and/orbind to activin include follistatin (FS), follistatin-related protein(FSRP), α₂-macroglobulin, Cerberus, and endoglin, which are describedbelow.

Nodal proteins have functions in mesoderm and endoderm induction andformation, as well as subsequent organization of axial structures suchas heart and stomach in early embryogenesis. It has been demonstratedthat dorsal tissue in a developing vertebrate embryo contributespredominantly to the axial structures of the notochord and pre-chordalplate while it recruits surrounding cells to form non-axial embryonicstructures. Nodal appears to signal through both type I and type IIreceptors and intracellular effectors known as Smad proteins. Recentstudies support the idea that ActRIIA and ActRIIB serve as type IIreceptors for Nodal (Sakuma et al., Genes Cells. 2002, 7:401-12). It issuggested that Nodal ligands interact with their co-factors (e.g.,cripto) to activate activin type I and type II receptors, whichphosphorylate Smad2. Nodal proteins are implicated in many eventscritical to the early vertebrate embryo, including mesoderm formation,anterior patterning, and left-right axis specification. Experimentalevidence has demonstrated that Nodal signaling activates pAR3-Lux, aluciferase reporter previously shown to respond specifically to activinand TGF-beta. However, Nodal is unable to induce pTlx2-Lux, a reporterspecifically responsive to bone morphogenetic proteins. Recent resultsprovide direct biochemical evidence that Nodal signaling is mediated byboth activin-TGF-beta pathway Smads, Smad2 and Smad3. Further evidencehas shown that the extracellular cripto protein is required for Nodalsignaling, making it distinct from activin or TGF-beta signaling.

Growth and Differentiation Factor-8 (GDF8) is also known as myostatin.GDF8 is a negative regulator of skeletal muscle mass. GDF8 is highlyexpressed in the developing and adult skeletal muscle. The GDF8 nullmutation in transgenic mice is characterized by a marked hypertrophy andhyperplasia of the skeletal muscle (McPherron et al., Nature, 1997,387:83-90). Similar increases in skeletal muscle mass are evident innaturally occurring mutations of GDF8 in cattle (Ashmore et al., 1974,Growth, 38:501-507; Swatland and Kieffer, J. Anim. Sci., 1994,38:752-757; McPherron and Lee, Proc. Natl. Acad. Sci. USA, 1997,94:12457-12461; and Kambadur et al., Genome Res., 1997, 7:910-915) and,strikingly, in humans (Schuelke et al., N Engl J Med 2004; 350:2682-8).Studies have also shown that muscle wasting associated withHIV-infection in humans is accompanied by increases in GDF8 proteinexpression (Gonzalez-Cadavid et al., PNAS, 1998, 95:14938-43). Inaddition, GDF8 can modulate the production of muscle-specific enzymes(e.g., creatine kinase) and modulate myoblast cell proliferation (WO00/43781). The GDF8 propeptide can noncovalently bind to the mature GDF8domain dimer, inactivating its biological activity (Miyazono et al.(1988) J. Biol. Chem., 263: 6407-6415; Wakefield et al. (1988) J. Biol.Chem., 263; 7646-7654; and Brown et al. (1990) Growth Factors, 3:35-43). Other proteins which bind to GDF8 or structurally relatedproteins and inhibit their biological activity include follistatin, andpotentially, follistatin-related proteins (Gamer et al. (1999) Dev.Biol., 208: 222-232).

Growth and Differentiation Factor-11 (GDF11), also known as BMP11, is asecreted protein (McPherron et al., 1999, Nat. Genet. 22: 260-264).GDF11 is expressed in the tail bud, limb bud, maxillary and mandibulararches, and dorsal root ganglia during mouse development (Nakashima etal., 1999, Mech. Dev. 80: 185-189). GDF11 plays a unique role inpatterning both mesodermal and neural tissues (Gamer et al., 1999, DevBiol., 208:222-32). GDF11 was shown to be a negative regulator ofchondrogenesis and myogenesis in developing chick limb (Gamer et al.,2001, Dev Biol. 229:407-20). The expression of GDF11 in muscle alsosuggests its role in regulating muscle growth in a similar way to GDF8.In addition, the expression of GDF11 in brain suggests that GDF11 mayalso possess activities that relate to the function of the nervoussystem. Interestingly, GDF11 was found to inhibit neurogenesis in theolfactory epithelium (Wu et al., 2003, Neuron. 37:197-207). Hence, GDF11may have in vitro and in vivo applications in the treatment of diseasessuch as muscle diseases and neurodegenerative diseases (e.g.,amyotrophic lateral sclerosis).

Bone morphogenetic protein (BMP7), also called osteogenic protein-1(OP-1), is well known to induce cartilage and bone formation. Inaddition, BMP7 regulates a wide array of physiological processes. Forexample, BMP7 may be the osteoinductive factor responsible for thephenomenon of epithelial osteogenesis. It is also found that BMP7 playsa role in calcium regulation and bone homeostasis. Like activin, BMP7binds to type II receptors, ActRIIA and IIB. However, BMP7 and activinrecruit distinct type I receptors into heteromeric receptor complexes.The major BMP7 type I receptor observed was ALK2, while activin boundexclusively to ALK4 (ActRIIB). BMP7 and activin elicited distinctbiological responses and activated different Smad pathways (Macias-Silvaet al., 1998, J Biol Chem. 273:25628-36).

In certain aspects, the present invention relates to the use of certainActRIIB polypeptides (e.g., soluble ActRIIB polypeptides) to antagonizethe signaling of ActRIIB ligands generally, in any process associatedwith ActRIIB activity. Optionally, ActRIIB polypeptides of the inventionmay antagonize one or more ligands of ActRIIB receptors, such asactivin, Nodal, GDF8, GDF11, and BMP7, and may therefore be useful inthe treatment of additional disorders.

Therefore, the present invention contemplates using ActRIIB polypeptidesin treating or preventing diseases or conditions that are associatedwith abnormal activity of an ActRIIB or an ActRIIB ligand. ActRIIB orActRIIB ligands are involved in the regulation of many criticalbiological processes. Due to their key functions in these processes,they may be desirable targets for therapeutic intervention. For example,ActRIIB polypeptides (e.g., soluble ActRIIB polypeptides) may be used totreat human or animal disorders or conditions. In particular, thepresent disclosure provides surprising evidence that antagonists ofActRIIB signaling, such as ActRIIB-Fc fusion proteins, may directlyaddress the underlying defect in dystrophin deficient patients byincreasing the level of utrophin present in the sarcolemma. Whileprevious publications have indicated that such agents may be useful forincreasing muscle mass and strength in muscular dystrophy patients,these data indicate that such agents may have a direct effect on musclefiber fragility in DMD and BMD patients. These disorders and conditionsare discussed below under “Exemplary Therapeutic Uses.”

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values.

Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The methods of the invention may include steps of comparing sequences toeach other, including wild-type sequence to one or more mutants(sequence variants). Such comparisons typically comprise alignments ofpolymer sequences, e.g., using sequence alignment programs and/oralgorithms that are well known in the art (for example, BLAST, FASTA andMEGALIGN, to name a few). The skilled artisan can readily appreciatethat, in such alignments, where a mutation contains a residue insertionor deletion, the sequence alignment will introduce a “gap” (typicallyrepresented by a dash, or “A”) in the polymer sequence not containingthe inserted or deleted residue.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

2. ActRIIB Polypeptides

In certain aspects, the invention relates to ActRIIB variantpolypeptides (e.g., soluble ActRIIB polypeptides). Optionally, thefragments, functional variants, and modified forms have similar or thesame biological activities of their corresponding wild-type ActRIIBpolypeptides. For example, an ActRIIB variant of the invention may bindto and inhibit function of an ActRIIB ligand (e.g., activin A, activinAB, activin B, Nodal, GDF8, GDF11 or BMP7). Optionally, an ActRIIBpolypeptide modulates growth of muscle. Examples of ActRIIB polypeptidesinclude human ActRIIB precursor polypeptide (SEQ ID NO: 2), and solublehuman ActRIIB polypeptides (e.g., SEQ ID NOs: 1, 5, and 12).

The disclosure identifies functionally active portions and variants ofActRIIB. Applicants have ascertained that an Fc fusion protein havingthe sequence disclosed by Hilden et al. (Blood. 1994 Apr. 15;83(8):2163-70), which has an Alanine at the position corresponding toamino acid 64 of SEQ ID NO: 2 (A64), has a relatively low affinity foractivin and GDF-11. By contrast, the same Fc fusion protein with anArginine at position 64 (R64) has an affinity for activin and GDF-11 inthe low nanomolar to high picomolar range. Therefore, a sequence with anR64 is used as the wild-type reference sequence for human ActRIIB inthis disclosure.

Attisano et al. (Cell. 1992 Jan. 10; 68(1):97-108) showed that adeletion of the proline knot at the C-terminus of the extracellulardomain of ActRIIB reduced the affinity of the receptor for activin. Datapresented here shows that an ActRIIB-Fc fusion protein containing aminoacids 20-119 of SEQ ID NO: 2, “ActRIIB(20-119)-Fc” has reduced bindingto GDF-11 and activin relative to an ActRIIB(20-134)-Fc, which includesthe proline knot region and the complete juxtamembrane domain. However,an ActRIIB(20-129)-Fc protein retains similar but somewhat reducedactivity relative to the wild type, even though the proline knot regionis disrupted. Thus, ActRIIB extracellular domains that stop at aminoacid 134, 133, 132, 131, 130 and 129 are all expected to be active, butconstructs stopping at 134 or 133 may be most active. Similarly,mutations at any of residues 129-134 are not expected to alter ligandbinding affinity by large margins. In support of this, mutations of P129and P130 do not substantially decrease ligand binding. Therefore, anActRIIB-Fc fusion protein may end as early as amino acid 109 (the finalcysteine), however, forms ending at or between 109 and 119 are expectedto have reduced ligand binding. Amino acid 119 is poorly conserved andso is readily altered or truncated. Forms ending at 128 or later retainligand binding activity. Forms ending at or between 119 and 127 willhave an intermediate binding ability. Any of these forms may bedesirable to use, depending on the clinical or experimental setting.

At the N-terminus of ActRIIB, it is expected that a protein beginning atamino acid 29 or before will retain ligand binding activity. Amino acid29 represents the initial cysteine. An alanine to asparagine mutation atposition 24 introduces an N-linked glycosylation sequence withoutsubstantially affecting ligand binding. This confirms that mutations inthe region between the signal cleavage peptide and the cysteinecross-linked region, corresponding to amino acids 20-29 are welltolerated. In particular, constructs beginning at position 20, 21, 22,23 and 24 will retain activity, and constructs beginning at positions25, 26, 27, 28 and 29 are also expected to retain activity. A constructbeginning at 22, 23, 24 or 25 will have the most activity.

Taken together, an active portion of ActRIIB comprises amino acids29-109 of SEQ ID NO: 2, and constructs may, for example, begin at aresidue corresponding to amino acids 20-29 and end at a positioncorresponding to amino acids 109-134. Other examples include constructsthat begin at a position from 20-29 or 21-29 and end at a position from119-134, 119-133 or 129-134, 129-133. Other examples include constructsthat begin at a position from 20-24 (or 21-24, or 22-25) and end at aposition from 109-134 (or 109-133), 119-134 (or 119-133) or 129-134 (or129-133). Variants within these ranges are also contemplated,particularly those having at least 80%, 85%, 90%, 95% or 99% identity tothe corresponding portion of SEQ ID NO: 2.

The disclosure includes the results of an analysis of composite ActRIIBstructures, shown in FIG. 5, demonstrating that the ligand bindingpocket is defined by residues Y31, N33, N35, L38 through T41, E47, E50,Q53 through K55, L57, H58, Y60, S62, K74, W78 through N83, Y85, R87,A92, and E94 through F101. At these positions, it is expected thatconservative mutations will be tolerated, although a K74A mutation iswell-tolerated, as are R40A, K55A, F82A and mutations at position L79.R40 is a K in Xenopus, indicating that basic amino acids at thisposition will be tolerated. Q53 is R in bovine ActRIIB and K in XenopusActRIIB, and therefore amino acids including R, K, Q, N and H will betolerated at this position. Thus, a general formula for an activeActRIIB variant protein is one that comprises amino acids 29-109, butoptionally beginning at a position ranging from 20-24 or 22-25 andending at a position ranging from 129-134, and comprising no more than1, 2, 5, 10 or 15 conservative amino acid changes in the ligand bindingpocket, and zero, one or more non-conservative alterations at positions40, 53, 55, 74, 79 and/or 82 in the ligand binding pocket. Such aprotein may retain greater than 80%, 90%, 95% or 99% sequence identityto the sequence of amino acids 29-109 of SEQ ID NO: 2. Sites outside thebinding pocket, at which variability may be particularly well tolerated,include the amino and carboxy termini of the extracellular domain (asnoted above), and positions 42-46 and 65-73. An asparagine to alaninealteration at position 65 (N65A) actually improves ligand binding in theA64 background, and is thus expected to have no detrimental effect onligand binding in the R64 background. This change probably eliminatesglycosylation at N65 in the A64 background, thus demonstrating that asignificant change in this region is likely to be tolerated. While anR64A change is poorly tolerated, R64K is well-tolerated, and thusanother basic residue, such as H may be tolerated at position 64.

ActRIIB is well-conserved across nearly all vertebrates, with largestretches of the extracellular domain conserved completely. Many of theligands that bind to ActRIIB are also highly conserved. Accordingly,comparisons of ActRIIB sequences from various vertebrate organismsprovide insights into residues that may be altered (FIG. 6). Therefore,an active, human ActRIIB variant may include one or more amino acids atcorresponding positions from the sequence of another vertebrate ActRIIB,or may include a residue that is similar to that in the human or othervertebrate sequence. The following examples illustrate this approach todefining an active ActRIIB variant. L46 is a valine in Xenopus ActRIIB,and so this position may be altered, and optionally may be altered toanother hydrophobic residue, such as V, I or F, or a non-polar residuesuch as A. E52 is a K in Xenopus, indicating that this site may betolerant of a wide variety of changes, including polar residues, such asE, D, K, R, H, S, T, P, G, Y and probably A. T93 is a K in Xenopus,indicating that a wide structural variation is tolerated at thisposition, with polar residues favored, such as S, K, R, E, D, H, G, P, Gand Y. F108 is a Y in Xenopus, and therefore Y or other hydrophobicgroup, such as I, V or L should be tolerated. E111 is K in Xenopus,indicating that charged residues will be tolerated at this position,including D, R, K and H, as well as Q and N. R112 is K in Xenopus,indicating that basic residues are tolerated at this position, includingR and H. A at position 119 is relatively poorly conserved, and appearsas Pin rodents and V in Xenopus, thus essentially any amino acid shouldbe tolerated at this position.

The disclosure demonstrates that the addition of a further N-linkedglycosylation site (N-X-S/T) increases the serum half-life of anActRIIB-Fc fusion protein, relative to the ActRIIB(R64)-Fc form. Byintroducing an asparagine at position 24 (A24N construct), an NXTsequence is created that confers a longer half-life. Other NX(T/S)sequences are found at 42-44 (NQS) and 65-67 (NSS), although the lattermay not be efficiently glycosylated with the R at position 64. N-X-S/Tsequences may be generally introduced at positions outside the ligandbinding pocket defined in FIG. 5. Particularly suitable sites for theintroduction of non-endogenous N-X-S/T sequences include amino acids20-29, 20-24, 22-25, 109-134, 120-134 or 129-134. N-X-S/T sequences mayalso be introduced into the linker between the ActRIIB sequence and theFc or other fusion component. Such a site may be introduced with minimaleffort by introducing an N in the correct position with respect to apre-existing S or T, or by introducing an S or T at a positioncorresponding to a pre-existing N. Thus, desirable alterations thatwould create an N-linked glycosylation site are: A24N, R64N, S67N(possibly combined with an N65A alteration), E106N, R112N, G120N, E123N,P129N, A132N, R112S and R112T. Any S that is predicted to beglycosylated may be altered to a T without creating an immunogenic site,because of the protection afforded by the glycosylation. Likewise, any Tthat is predicted to be glycosylated may be altered to an S. Thus thealterations S67T and S44T are contemplated. Likewise, in an A24Nvariant, an S26T alteration may be used. Accordingly, an ActRIIB variantmay include one or more additional, non-endogenous N-linkedglycosylation consensus sequences.

Position L79 may be altered to confer altered activin—myostatin (GDF-11)binding properties. L79A or L79P reduces GDF-11 binding to a greaterextent than activin binding. L79E or L79D retains GDF-11 binding.Remarkably, the L79E and L79D variants have greatly reduced activinbinding. In vivo experiments indicate that these non-activin receptorsretain significant ability to increase muscle mass but show decreasedeffects on other tissues. These data demonstrate the desirability andfeasibility for obtaining polypeptides with reduced effects on activin.

The variations described may be combined in various ways. Additionally,the results of mutagenesis program described herein indicate that thereare amino acid positions in ActRIIB that are often beneficial toconserve. These include position 64 (basic amino acid), position 80(acidic or hydrophobic amino acid), position 78 (hydrophobic, andparticularly tryptophan), position 37 (acidic, and particularly asparticor glutamic acid), position 56 (basic amino acid), position 60(hydrophobic amino acid, particularly phenylalanine or tyrosine). Thus,in each of the variants disclosed herein, the disclosure provides aframework of amino acids that may be conserved. Other positions that maybe desirable to conserve are as follows: position 52 (acidic aminoacid), position 55 (basic amino acid), position 81 (acidic), 98 (polaror charged, particularly E, D, R or K).

In certain embodiments, isolated fragments of the ActRIIB polypeptidescan be obtained by screening polypeptides recombinantly produced fromthe corresponding fragment of the nucleic acid encoding an ActRIIBpolypeptide (e.g., SEQ ID NOs: 3 and 4). In addition, fragments can bechemically synthesized using techniques known in the art such asconventional Merrifield solid phase f-Moc or t-Boc chemistry. Thefragments can be produced (recombinantly or by chemical synthesis) andtested to identify those peptidyl fragments that can function, forexample, as antagonists (inhibitors) or agonists (activators) of anActRIIB protein or an ActRIIB ligand.

In certain embodiments, a functional variant of the ActRIIB polypeptideshas an amino acid sequence that is at least 75% identical to an aminoacid sequence selected from SEQ ID NOs: 1, 2, 5, 12, and 23. In certaincases, the functional variant has an amino acid sequence at least 80%,85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequenceselected from SEQ ID NOs: 1, 2, 5, 12, and 23.

In certain embodiments, the present invention contemplates makingfunctional variants by modifying the structure of an ActRIIB polypeptidefor such purposes as enhancing therapeutic efficacy, or stability (e.g.,ex vivo shelf life and resistance to proteolytic degradation in vivo).Modified ActRIIB polypeptides can also be produced, for instance, byamino acid substitution, deletion, or addition. For instance, it isreasonable to expect that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar replacement of an amino acid with a structurallyrelated amino acid (e.g., conservative mutations) will not have a majoreffect on the biological activity of the resulting molecule.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Whether a change inthe amino acid sequence of an ActRIIB polypeptide results in afunctional homolog can be readily determined by assessing the ability ofthe variant ActRIIB polypeptide to produce a response in cells in afashion similar to the wild-type ActRIIB polypeptide, or to bind to oneor more ligands, such as activin, GDF-11 or myostatin in a fashionsimilar to wild type.

In certain specific embodiments, the present invention contemplatesmaking mutations in the extracellular domain (also referred to asligand-binding domain) of an ActRIIB polypeptide such that the variant(or mutant) ActRIIB polypeptide has altered ligand-binding activities(e.g., binding affinity or binding specificity). In certain cases, suchvariant ActRIIB polypeptides have altered (elevated or reduced) bindingaffinity for a specific ligand. In other cases, the variant ActRIIBpolypeptides have altered binding specificity for their ligands.

For example, the disclosure provides variant ActRIIB polypeptides thatpreferentially bind to GDF8/GDF11 relative to activins. The disclosurefurther establishes the desirability of such polypeptides for reducingoff-target effects, although such selective variants may be lessdesirable for the treatment of severe diseases where very large gains inmuscle mass may be needed for therapeutic effect and where some level ofoff-target effect is acceptable. For example, amino acid residues of theActRIIB protein, such as E39, K55, Y60, K74, W78, D80, and F101, are inthe ligand-binding pocket and mediate binding to its ligands such asactivin and GDF8. Thus, the present invention provides an alteredligand-binding domain (e.g., GDF8-binding domain) of an ActRIIBreceptor, which comprises one or more mutations at those amino acidresidues. Optionally, the altered ligand-binding domain can haveincreased selectivity for a ligand such as GDF8 relative to a wild-typeligand-binding domain of an ActRIIB receptor. To illustrate, thesemutations increase the selectivity of the altered ligand-binding domainfor GDF8 over activin. Optionally, the altered ligand-binding domain hasa ratio of K_(d) for activin binding to K_(d) for GDF8 binding that isat least 2, 5, 10, or even 100 fold greater relative to the ratio forthe wild-type ligand-binding domain. Optionally, the alteredligand-binding domain has a ratio of IC₅₀ for inhibiting activin to IC₅₀for inhibiting GDF8 that is at least 2, 5, 10, or even 100 fold greaterrelative to the wild-type ligand-binding domain. Optionally, the alteredligand-binding domain inhibits GDF8 with an IC₅₀ at least 2, 5, 10, oreven 100 times less than the IC₅₀ for inhibiting activin.

As a specific example, the positively-charged amino acid residue Asp(D80) of the ligand-binding domain of ActRIIB can be mutated to adifferent amino acid residue such that the variant ActRIIB polypeptidepreferentially binds to GDF8, but not activin. Preferably, the D80residue is changed to an amino acid residue selected from the groupconsisting of: a uncharged amino acid residue, a negative amino acidresidue, and a hydrophobic amino acid residue. As a further specificexample, the hydrophobic residue L79 can be altered to the acidic aminoacids aspartic acid or glutamic acid to greatly reduce activin bindingwhile retaining GDF11 binding. As will be recognized by one of skill inthe art, most of the described mutations, variants or modifications maybe made at the nucleic acid level or, in some cases, by posttranslational modification or chemical synthesis. Such techniques arewell known in the art.

In certain embodiments, the present invention contemplates specificmutations of the ActRIIB polypeptides so as to alter the glycosylationof the polypeptide. Exemplary glycosylation sites in ActRIIBpolypeptides are illustrated in FIG. 2. Such mutations may be selectedso as to introduce or eliminate one or more glycosylation sites, such asO-linked or N-linked glycosylation sites. Asparagine-linkedglycosylation recognition sites generally comprise a tripeptidesequence, asparagine-X-threonine (where “X” is any amino acid) which isspecifically recognized by appropriate cellular glycosylation enzymes.The alteration may also be made by the addition of, or substitution by,one or more serine or threonine residues to the sequence of thewild-type ActRIIB polypeptide (for O-linked glycosylation sites). Avariety of amino acid substitutions or deletions at one or both of thefirst or third amino acid positions of a glycosylation recognition site(and/or amino acid deletion at the second position) results innon-glycosylation at the modified tripeptide sequence. Another means ofincreasing the number of carbohydrate moieties on an ActRIIB polypeptideis by chemical or enzymatic coupling of glycosides to the ActRIIBpolypeptide. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine; (b) free carboxyl groups; (c)free sulfhydryl groups such as those of cysteine; (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline; (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan; or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330 published Sep. 11, 1987, and in Aplin andWriston (1981) CRC Crit. Rev. Biochem., pp. 259-306, incorporated byreference herein. Removal of one or more carbohydrate moieties presenton an ActRIIB polypeptide may be accomplished chemically and/orenzymatically. Chemical deglycosylation may involve, for example,exposure of the ActRIIB polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Chemical deglycosylation is further described byHakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge etal. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydratemoieties on ActRIIB polypeptides can be achieved by the use of a varietyof endo- and exo-glycosidases as described by Thotakura et al. (1987)Meth. Enzymol. 138:350. The sequence of an ActRIIB polypeptide may beadjusted, as appropriate, depending on the type of expression systemused, as mammalian, yeast, insect and plant cells may all introducediffering glycosylation patterns that can be affected by the amino acidsequence of the peptide. In general, ActRIIB proteins for use in humanswill be expressed in a mammalian cell line that provides properglycosylation, such as HEK293 or CHO cell lines, although othermammalian expression cell lines are expected to be useful as well.

This disclosure further contemplates a method of generating variants,particularly sets of combinatorial variants of an ActRIIB polypeptide,including, optionally, truncation variants; pools of combinatorialmutants are especially useful for identifying functional variantsequences. The purpose of screening such combinatorial libraries may beto generate, for example, ActRIIB polypeptide variants which havealtered properties, such as altered pharmacokinetics, or altered ligandbinding. A variety of screening assays are provided below, and suchassays may be used to evaluate variants. For example, an ActRIIBpolypeptide variant may be screened for ability to bind to an ActRIIBpolypeptide, to prevent binding of an ActRIIB ligand to an ActRIIBpolypeptide.

The activity of an ActRIIB polypeptide or its variants may also betested in a cell-based or in vivo assay. For example, the effect of anActRIIB polypeptide variant on the expression of genes involved in boneproduction in an osteoblast or precursor may be assessed. This may, asneeded, be performed in the presence of one or more recombinant ActRIIBligand protein (e.g., BMP7), and cells may be transfected so as toproduce an ActRIIB polypeptide and/or variants thereof, and optionally,an ActRIIB ligand. Likewise, an ActRIIB polypeptide may be administeredto a mouse or other animal, and one or more bone properties, such asdensity or volume may be assessed. The healing rate for bone fracturesmay also be evaluated. Similarly, the activity of an ActRIIB polypeptideor its variants may be tested in muscle cells, adipocytes, and neuronalcells for any effect on growth of these cells, for example, by theassays as described below. Such assays are well known and routine in theart. A SMAD-responsive reporter gene may be used in such cell lines tomonitor effects on downstream signaling.

Combinatorically-derived variants can be generated which have aselective potency relative to a naturally occurring ActRIIB polypeptide.Such variant proteins, when expressed from recombinant DNA constructs,can be used in gene therapy protocols. Likewise, mutagenesis can giverise to variants which have intracellular half-lives dramaticallydifferent than the corresponding wild-type ActRIIB polypeptide. Forexample, the altered protein can be rendered either more stable or lessstable to proteolytic degradation or other processes which result indestruction of, or otherwise inactivation of a native ActRIIBpolypeptide. Such variants, and the genes which encode them, can beutilized to alter ActRIIB polypeptide levels by modulating the half-lifeof the ActRIIB polypeptides. For instance, a short half-life can giverise to more transient biological effects and, when part of an inducibleexpression system, can allow tighter control of recombinant ActRIIBpolypeptide levels within the cell.

In certain embodiments, the ActRIIB polypeptides of the invention mayfurther comprise post-translational modifications in addition to anythat are naturally present in the ActRIIB polypeptides. Suchmodifications include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. As a result, the modified ActRIIB polypeptides may containnon-amino acid elements, such as polyethylene glycols, lipids, poly- ormono-saccharide, and phosphates. Effects of such non-amino acid elementson the functionality of a ActRIIB polypeptide may be tested as describedherein for other ActRIIB polypeptide variants. When an ActRIIBpolypeptide is produced in cells by cleaving a nascent form of theActRIIB polypeptide, post-translational processing may also be importantfor correct folding and/or function of the protein. Different cells(such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have specificcellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the ActRIIB polypeptides.

In certain aspects, functional variants or modified forms of the ActRIIBpolypeptides include fusion proteins having at least a portion of theActRIIB polypeptides and one or more fusion domains. Well known examplesof such fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, an immunoglobulin heavy chain constant region (e.g., an Fc),maltose binding protein (MBP), or human serum albumin. A fusion domainmay be selected so as to confer a desired property. For example, somefusion domains are particularly useful for isolation of the fusionproteins by affinity chromatography. For the purpose of affinitypurification, relevant matrices for affinity chromatography, such asglutathione-, amylase-, and nickel- or cobalt-conjugated resins areused. Many of such matrices are available in “kit” form, such as thePharmacia GST purification system and the QIAexpress™ system (Qiagen)useful with (HIS₆ (SEQ ID NO: 28)) fusion partners. As another example,a fusion domain may be selected so as to facilitate detection of theActRIIB polypeptides. Examples of such detection domains include thevarious fluorescent proteins (e.g., GFP) as well as “epitope tags,”which are usually short peptide sequences for which a specific antibodyis available. Well known epitope tags for which specific monoclonalantibodies are readily available include FLAG, influenza virushaemagglutinin (HA), and c-myc tags. In some cases, the fusion domainshave a protease cleavage site, such as for Factor Xa or Thrombin, whichallows the relevant protease to partially digest the fusion proteins andthereby liberate the recombinant proteins therefrom. The liberatedproteins can then be isolated from the fusion domain by subsequentchromatographic separation. In certain preferred embodiments, an ActRIIBpolypeptide is fused with a domain that stabilizes the ActRIIBpolypeptide in vivo (a “stabilizer” domain). By “stabilizing” is meantanything that increases serum half life, regardless of whether this isbecause of decreased destruction, decreased clearance by the kidney, orother pharmacokinetic effect. Fusions with the Fc portion of animmunoglobulin are known to confer desirable pharmacokinetic propertieson a wide range of proteins. Likewise, fusions to human serum albumincan confer desirable properties. Other types of fusion domains that maybe selected include multimerizing (e.g., dimerizing, tetramerizing)domains and functional domains (that confer an additional biologicalfunction, such as further stimulation of muscle growth).

As a specific example, the present invention provides a fusion proteinas a GDF8 antagonist which comprises an extracellular (e.g.,GDF8-binding) domain fused to an Fc domain (e.g., SEQ ID NO: 13).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK*

Preferably, the Fc domain has one or more mutations at residues such asAsp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc domainhaving one or more of these mutations (e.g., Asp-265 mutation) hasreduced ability of binding to the Fcγ receptor relative to a wildtype Fcdomain. In other cases, the mutant Fc domain having one or more of thesemutations (e.g., Asn-434 mutation) has increased ability of binding tothe MHC class I-related Fc-receptor (FcRN) relative to a wildtype Fcdomain.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, an ActRIIB polypeptide may be placedC-terminal to a heterologous domain, or, alternatively, a heterologousdomain may be placed C-terminal to an ActRIIB polypeptide. The ActRIIBpolypeptide domain and the heterologous domain need not be adjacent in afusion protein, and additional domains or amino acid sequences may beincluded C- or N-terminal to either domain or between the domains.

In certain embodiments, the ActRIIB polypeptides of the presentinvention contain one or more modifications that are capable ofstabilizing the ActRIIB polypeptides. For example, such modificationsenhance the in vitro half life of the ActRIIB polypeptides, enhancecirculatory half life of the ActRIIB polypeptides or reducingproteolytic degradation of the ActRIIB polypeptides. Such stabilizingmodifications include, but are not limited to, fusion proteins(including, for example, fusion proteins comprising an ActRIIBpolypeptide and a stabilizer domain), modifications of a glycosylationsite (including, for example, addition of a glycosylation site to anActRIIB polypeptide), and modifications of carbohydrate moiety(including, for example, removal of carbohydrate moieties from anActRIIB polypeptide). In the case of fusion proteins, an ActRIIBpolypeptide is fused to a stabilizer domain such as an IgG molecule(e.g., an Fc domain). As used herein, the term “stabilizer domain” notonly refers to a fusion domain (e.g., Fc) as in the case of fusionproteins, but also includes nonproteinaceous modifications such as acarbohydrate moiety, or nonproteinaceous polymer, such as polyethyleneglycol.

In certain embodiments, the present invention makes available isolatedand/or purified forms of the ActRIIB polypeptides, which are isolatedfrom, or otherwise substantially free of, other proteins.

In certain embodiments, ActRIIB polypeptides (unmodified or modified) ofthe invention can be produced by a variety of art-known techniques. Forexample, such ActRIIB polypeptides can be synthesized using standardprotein chemistry techniques such as those described in Bodansky, M.Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) andGrant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman andCompany, New York (1992). In addition, automated peptide synthesizersare commercially available (e.g., Advanced ChemTech Model 396;Milligen/Biosearch 9600). Alternatively, the ActRIIB polypeptides,fragments or variants thereof may be recombinantly produced usingvarious expression systems (e.g., E. coli, Chinese Hamster Ovary cells,COS cells, baculovirus) as is well known in the art (also see below). Ina further embodiment, the modified or unmodified ActRIIB polypeptidesmay be produced by digestion of naturally occurring or recombinantlyproduced full-length ActRIIB polypeptides by using, for example, aprotease, e.g., trypsin, thermolysin, chymotrypsin, pepsin, or pairedbasic amino acid converting enzyme (PACE). Computer analysis (using acommercially available software, e.g., MacVector, Omega, PCGene,Molecular Simulation, Inc.) can be used to identify proteolytic cleavagesites. Alternatively, such ActRIIB polypeptides may be produced fromnaturally occurring or recombinantly produced full-length ActRIIBpolypeptides such as standard techniques known in the art, such as bychemical cleavage (e.g., cyanogen bromide, hydroxylamine).

3. Nucleic Acids Encoding ActRIIB Polypeptides

In certain aspects, the invention provides isolated and/or recombinantnucleic acids encoding any of the ActRIIB polypeptides (e.g., solubleActRIIB polypeptides), including any of the variants disclosed herein.For example, SEQ ID NO: 4 encodes a naturally occurring ActRIIBprecursor polypeptide (FIG. 4), while SEQ ID NO: 3 encodes a solubleActRIIB polypeptide (FIG. 3). The subject nucleic acids may besingle-stranded or double stranded. Such nucleic acids may be DNA or RNAmolecules. These nucleic acids are may be used, for example, in methodsfor making ActRIIB polypeptides or as direct therapeutic agents (e.g.,in a gene therapy approach).

In certain aspects, the subject nucleic acids encoding ActRIIBpolypeptides are further understood to include nucleic acids that arevariants of SEQ ID NO: 3. Variant nucleotide sequences include sequencesthat differ by one or more nucleotide substitutions, additions ordeletions, such as allelic variants; and will, therefore, include codingsequences that differ from the nucleotide sequence of the codingsequence designated in SEQ ID NO: 4.

In certain embodiments, the invention provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to SEQ ID NO: 3. One of ordinary skill in the artwill appreciate that nucleic acid sequences complementary to SEQ ID NO:3, and variants of SEQ ID NO: 3 are also within the scope of thisinvention. In further embodiments, the nucleic acid sequences of theinvention can be isolated, recombinant, and/or fused with a heterologousnucleotide sequence, or in a DNA library.

In other embodiments, nucleic acids of the invention also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequence designated in SEQ ID NO: 3, complement sequenceof SEQ ID NO: 3, or fragments thereof. As discussed above, one ofordinary skill in the art will understand readily that appropriatestringency conditions which promote DNA hybridization can be varied. Oneof ordinary skill in the art will understand readily that appropriatestringency conditions which promote DNA hybridization can be varied. Forexample, one could perform the hybridization at 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or temperature or saltconcentration may be held constant while the other variable is changed.In one embodiment, the invention provides nucleic acids which hybridizeunder low stringency conditions of 6×SSC at room temperature followed bya wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NO: 3 due to degeneracy in the genetic code are also withinthe scope of the invention. For example, a number of amino acids aredesignated by more than one triplet. Codons that specify the same aminoacid, or synonyms (for example, CAU and CAC are synonyms for histidine)may result in “silent” mutations which do not affect the amino acidsequence of the protein. However, it is expected that DNA sequencepolymorphisms that do lead to changes in the amino acid sequences of thesubject proteins will exist among mammalian cells. One skilled in theart will appreciate that these variations in one or more nucleotides (upto about 3-5% of the nucleotides) of the nucleic acids encoding aparticular protein may exist among individuals of a given species due tonatural allelic variation. Any and all such nucleotide variations andresulting amino acid polymorphisms are within the scope of thisinvention.

In certain embodiments, the recombinant nucleic acids of the inventionmay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects of the invention, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding an ActRIIB polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the ActRIIB polypeptide. Accordingly,the term regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding an ActRIIB polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the trp system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda, thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid of the invention can be produced by ligatingthe cloned gene, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells (yeast, avian,insect or mammalian), or both. Expression vehicles for production of arecombinant ActRIIB polypeptide include plasmids and other vectors. Forinstance, suitable vectors include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 1989) Chapters 16 and 17. In some instances, it may bedesirable to express the recombinant polypeptides by the use of abaculovirus expression system. Examples of such baculovirus expressionsystems include pVL-derived vectors (such as pVL1392, pVL1393 andpVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derivedvectors (such as the ß-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject ActRIIB polypeptides in CHO cells, such as a Pcmv-Scriptvector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen,Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As willbe apparent, the subject gene constructs can be used to cause expressionof the subject ActRIIB polypeptides in cells propagated in culture,e.g., to produce proteins, including fusion proteins or variantproteins, for purification.

This invention also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NO: 4) forone or more of the subject ActRIIB polypeptide. The host cell may be anyprokaryotic or eukaryotic cell. For example, an ActRIIB polypeptide ofthe invention may be expressed in bacterial cells such as E. coli,insect cells (e.g., using a baculovirus expression system), yeast, ormammalian cells. Other suitable host cells are known to those skilled inthe art.

Accordingly, the present invention further pertains to methods ofproducing the subject ActRIIB polypeptides. For example, a host celltransfected with an expression vector encoding an ActRIIB polypeptidecan be cultured under appropriate conditions to allow expression of theActRIIB polypeptide to occur. The ActRIIB polypeptide may be secretedand isolated from a mixture of cells and medium containing the ActRIIBpolypeptide. Alternatively, the ActRIIB polypeptide may be retainedcytoplasmically or in a membrane fraction and the cells harvested, lysedand the protein isolated. A cell culture includes host cells, media andother byproducts. Suitable media for cell culture are well known in theart. The subject ActRIIB polypeptides can be isolated from cell culturemedium, host cells, or both, using techniques known in the art forpurifying proteins, including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for particularepitopes of the ActRIIB polypeptides. In a preferred embodiment, theActRIIB polypeptide is a fusion protein containing a domain whichfacilitates its purification.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant ActRIIBpolypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified ActRIIB polypeptide (e.g., seeHochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al.,PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

4. Antibodies

Another aspect of the invention pertains to antibodies. An antibody thatis specifically reactive with an ActRIIB polypeptide (e.g., a solubleActRIIB polypeptide) and which binds competitively with the ActRIIBpolypeptide may be used as an antagonist of ActRIIB polypeptideactivities. For example, by using immunogens derived from an ActRIIBpolypeptide, anti-protein/anti-peptide antisera or monoclonal antibodiescan be made by standard protocols (see, for example, Antibodies: ALaboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press:1988)). A mammal, such as a mouse, a hamster or rabbit can be immunizedwith an immunogenic form of the ActRIIB polypeptide, an antigenicfragment which is capable of eliciting an antibody response, or a fusionprotein. Techniques for conferring immunogenicity on a protein orpeptide include conjugation to carriers or other techniques well knownin the art. An immunogenic portion of an ActRIIB polypeptide can beadministered in the presence of adjuvant. The progress of immunizationcan be monitored by detection of antibody titers in plasma or serum.Standard ELISA or other immunoassays can be used with the immunogen asantigen to assess the levels of antibodies.

Following immunization of an animal with an antigenic preparation of anActRIIB polypeptide, antisera can be obtained and, if desired,polyclonal antibodies can be isolated from the serum. To producemonoclonal antibodies, antibody-producing cells (lymphocytes) can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with an ActRIIBpolypeptide and monoclonal antibodies isolated from a culture comprisingsuch hybridoma cells.

The term “antibody” as used herein is intended to include fragmentsthereof which are also specifically reactive with a subject ActRIIBpolypeptide. Antibodies can be fragmented using conventional techniquesand the fragments screened for utility in the same manner as describedabove for whole antibodies. For example, F(ab)₂ fragments can begenerated by treating antibody with pepsin. The resulting F(ab)₂fragment can be treated to reduce disulfide bridges to produce Fabfragments. The antibody of the present invention is further intended toinclude bispecific, single-chain, and chimeric and humanized moleculeshaving affinity for an ActRIIB polypeptide conferred by at least one CDRregion of the antibody. In preferred embodiments, the antibody furthercomprises a label attached thereto and able to be detected (e.g., thelabel can be a radioisotope, fluorescent compound, enzyme or enzymeco-factor).

In certain preferred embodiments, an antibody of the invention is amonoclonal antibody, and in certain embodiments, the invention makesavailable methods for generating novel antibodies. For example, a methodfor generating a monoclonal antibody that binds specifically to anActRIIB polypeptide may comprise administering to a mouse an amount ofan immunogenic composition comprising the ActRIIB polypeptide effectiveto stimulate a detectable immune response, obtaining antibody-producingcells (e.g., cells from the spleen) from the mouse and fusing theantibody-producing cells with myeloma cells to obtain antibody-producinghybridomas, and testing the antibody-producing hybridomas to identify ahybridoma that produces a monocolonal antibody that binds specificallyto the ActRIIB polypeptide. Once obtained, a hybridoma can be propagatedin a cell culture, optionally in culture conditions where thehybridoma-derived cells produce the monoclonal antibody that bindsspecifically to the ActRIIB polypeptide. The monoclonal antibody may bepurified from the cell culture.

The adjective “specifically reactive with” as used in reference to anantibody is intended to mean, as is generally understood in the art,that the antibody is sufficiently selective between the antigen ofinterest (e.g., an ActRIIB polypeptide) and other antigens that are notof interest that the antibody is useful for, at minimum, detecting thepresence of the antigen of interest in a particular type of biologicalsample. In certain methods employing the antibody, such as therapeuticapplications, a higher degree of specificity in binding may bedesirable. Monoclonal antibodies generally have a greater tendency (ascompared to polyclonal antibodies) to discriminate effectively betweenthe desired antigens and cross-reacting polypeptides. One characteristicthat influences the specificity of an antibody:antigen interaction isthe affinity of the antibody for the antigen. Although the desiredspecificity may be reached with a range of different affinities,generally preferred antibodies will have an affinity (a dissociationconstant) of about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or less.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, if an antibody is to be used for bindingan antigen in solution, it may be desirable to test solution binding. Avariety of different techniques are available for testing interactionbetween antibodies and antigens to identify particularly desirableantibodies. Such techniques include ELISAs, surface plasmon resonancebinding assays (e.g., the Biacore binding assay, Bia-core AB, Uppsala,Sweden), sandwich assays (e.g., the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Md.), western blots,immunoprecipitation assays, and immunohistochemistry.

In certain aspects, the disclosure provides antibodies that bind to asoluble ActRIIB polypeptide. Such antibodies may be generated much asdescribed above, using a soluble ActRIIB polypeptide or fragment thereofas an antigen. Antibodies of this type can be used, e.g., to detectActRIIB polypeptides in biological samples and/or to monitor solubleActRIIB polypeptide levels in an individual. In certain cases, anantibody that specifically binds to a soluble ActRIIB polypeptide can beused to modulate activity of an ActRIIB polypeptide and/or an ActRIIBligand, thereby regulating (promoting or inhibiting) growth of tissues,such as bone, cartilage, muscle, fat, and neurons, or increasingsarcolemmal utrophin.

5. Screening Assays

In certain aspects, the present invention relates to the use of thesubject ActRIIB polypeptides (e.g., soluble ActRIIB polypeptides) toidentify compounds (agents) which are agonist or antagonists of theActRIIB polypeptides. Compounds identified through this screening can betested in tissues such as bone, cartilage, muscle, fat, and/or neurons,to assess their ability to modulate tissue growth in vitro. Optionally,these compounds can further be tested in animal models to assess theirability to modulate tissue growth in vivo.

There are numerous approaches to screening for therapeutic agents formodulating tissue growth by targeting the ActRIIB polypeptides. Incertain embodiments, high-throughput screening of compounds can becarried out to identify agents that perturb ActRIIB-mediated effects ongrowth of bone, cartilage, muscle, fat, and/or neurons. In certainembodiments, the assay is carried out to screen and identify compoundsthat specifically inhibit or reduce binding of an ActRIIB polypeptide toits binding partner, such as an ActRIIB ligand (e.g., activin, Nodal,GDF8, GDF11 or BMP7). Alternatively, the assay can be used to identifycompounds that enhance binding of an ActRIIB polypeptide to its bindingprotein such as an ActRIIB ligand. In a further embodiment, thecompounds can be identified by their ability to interact with an ActRIIBpolypeptide.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof tissue growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. In aspecific embodiment, the test agent is a small organic molecule having amolecular weight of less than about 2,000 daltons.

The test compounds of the invention can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S transferase (GST),photoactivatible crosslinkers or any combinations thereof.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between an ActRIIBpolypeptide and its binding protein (e.g., an ActRIIB ligand).

Merely to illustrate, in an exemplary screening assay of the presentinvention, the compound of interest is contacted with an isolated andpurified ActRIIB polypeptide which is ordinarily capable of binding toan ActRIIB ligand, as appropriate for the intention of the assay. To themixture of the compound and ActRIIB polypeptide is then added acomposition containing an ActRIIB ligand. Detection and quantificationof ActRIIB/ActRIIB ligand complexes provides a means for determining thecompound's efficacy at inhibiting (or potentiating) complex formationbetween the ActRIIB polypeptide and its binding protein. The efficacy ofthe compound can be assessed by generating dose response curves fromdata obtained using various concentrations of the test compound.Moreover, a control assay can also be performed to provide a baselinefor comparison. For example, in a control assay, isolated and purifiedActRIIB ligand is added to a composition containing the ActRIIBpolypeptide, and the formation of ActRIIB/ActRIIB ligand complex isquantitated in the absence of the test compound. It will be understoodthat, in general, the order in which the reactants may be admixed can bevaried, and can be admixed simultaneously. Moreover, in place ofpurified proteins, cellular extracts and lysates may be used to render asuitable cell-free assay system.

Complex formation between the ActRIIB polypeptide and its bindingprotein may be detected by a variety of techniques. For instance,modulation of the formation of complexes can be quantitated using, forexample, detectably labeled proteins such as radiolabeled (e.g., ³²P,³⁵S, ¹⁴C or ³H), fluorescently labeled (e.g., FITC), or enzymaticallylabeled ActRIIB polypeptide or its binding protein, by immunoassay, orby chromatographic detection.

In certain embodiments, the present invention contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between an ActRIIB polypeptide and its bindingprotein. Further, other modes of detection, such as those based onoptical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No.5,677,196), surface plasmon resonance (SPR), surface charge sensors, andsurface force sensors, are compatible with many embodiments of theinvention.

Moreover, the present invention contemplates the use of an interactiontrap assay, also known as the “two hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between an ActRIIB polypeptideand its binding protein. See for example, U.S. Pat. No. 5,283,317;Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; andIwabuchi et al. (1993) Oncogene 8:1693-1696). In a specific embodiment,the present invention contemplates the use of reverse two hybrid systemsto identify compounds (e.g., small molecules or peptides) thatdissociate interactions between an ActRIIB polypeptide and its bindingprotein. See for example, Vidal and Legrain, (1999) Nucleic Acids Res27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; andU.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368.

In certain embodiments, the subject compounds are identified by theirability to interact with an ActRIIB polypeptide of the invention. Theinteraction between the compound and the ActRIIB polypeptide may becovalent or non-covalent. For example, such interaction can beidentified at the protein level using in vitro biochemical methods,including photo-crosslinking, radiolabeled ligand binding, and affinitychromatography (Jakoby W B et al., 1974, Methods in Enzymology 46: 1).In certain cases, the compounds may be screened in a mechanism basedassay, such as an assay to detect compounds which bind to an ActRIIBpolypeptide. This may include a solid phase or fluid phase bindingevent. Alternatively, the gene encoding an ActRIIB polypeptide can betransfected with a reporter system (e.g., β-galactosidase, luciferase,or green fluorescent protein) into a cell and screened against thelibrary preferably by a high throughput screening or with individualmembers of the library. Other mechanism based binding assays may beused, for example, binding assays which detect changes in free energy.Binding assays can be performed with the target fixed to a well, bead orchip or captured by an immobilized antibody or resolved by capillaryelectrophoresis. The bound compounds may be detected usually usingcolorimetric or fluorescence or surface plasmon resonance.

In certain aspects, the present invention provides methods and agentsfor stimulating muscle growth and increasing muscle mass, for example,by antagonizing functions of an ActRIIB polypeptide and/or an ActRIIBligand. Therefore, any compound identified can be tested in whole cellsor tissues, in vitro or in vivo, to confirm their ability to modulatemuscle growth or alter utrophin levels at the sarcolemma. Variousmethods known in the art can be utilized for this purpose. For example,methods of the invention are performed such that the signal transductionthrough an ActRIIB protein activated by binding to an ActRIIB ligand(e.g., GDF8) has been reduced or inhibited. It will be recognized thatthe growth of muscle tissue in the organism would result in an increasedmuscle mass in the organism as compared to the muscle mass of acorresponding organism (or population of organisms) in which the signaltransduction through an ActRIIB protein had not been so effected.

For example, the effect of the ActRIIB polypeptides or test compounds onmuscle cell growth/proliferation can be determined by measuring geneexpression of Pax-3 and Myf-5 which are associated with proliferation ofmyogenic cells, and gene expression of MyoD which is associated withmuscle differentiation (e.g., Amthor et al., Dev Biol. 2002,251:241-57). It is known that GDF8 down-regulates gene expression ofPax-3 and Myf-5, and prevents gene expression of MyoD. The ActRIIBpolypeptides or test compounds are expected to antagonize this activityof GDF8. Another example of cell-based assays includes measuring theproliferation of myoblasts such as C(2)C(12) myoblasts in the presenceof the ActRIIB polypeptides or test compounds (e.g., Thomas et al., JBiol Chem. 2000, 275:40235-43).

The present invention also contemplates in vivo or ex vivo assays tomeasure muscle mass and strength. For example, Whittemore et al.(Biochem Biophys Res Commun. 2003, 300:965-71) disclose a method ofmeasuring increased skeletal muscle mass and increased grip strength inmice. Optionally, this method can be used to determine therapeuticeffects of test compounds (e.g., ActRIIB polypeptides) on musclediseases or conditions, for example those diseases for which muscle massis limiting. Moreover, the mechanical response of a muscle to repeatedcontraction can be used to assess the physiological integrity of thatmuscle after therapeutic intervention. For example, mouse models ofmuscular dystrophy typically display an excessive drop in maximaltetanic force after a series of eccentric contractions, during whichmuscle fibers lengthen as they exert force (contract). A smaller drop inforce after administration of a therapeutic agent (e.g., ActRIIBpolypeptide) can indicate beneficial effects on sarcolemmal integrity.Thus, Krag et al. (2004, Proc Natl Acad Sci USA 101:13856-13860)disclose a method for conducting such assays with isolated muscles exvivo. Alternatively, Blaauw et al. (2008, Hum Mol Genet 17:3686-3696)disclose a method for such testing in vivo.

It is understood that the screening assays of the present inventionapply to not only the subject ActRIIB polypeptides and variants of theActRIIB polypeptides, but also any test compounds including agonists andantagonist of the ActRIIB polypeptides. Further, these screening assaysare useful for drug target verification and quality control purposes.

6. Exemplary Therapeutic Uses

In certain embodiments, compositions (e.g., ActRIIB polypeptides) of thepresent invention can be used for treating or preventing a disease orcondition that is associated with abnormal activity of an ActRIIBpolypeptide and/or an ActRIIB ligand (e.g., GDF8). These diseases,disorders or conditions are generally referred to herein as“ActRIIB-associated conditions.” In certain embodiments, the presentinvention provides methods of treating or preventing a disease,disorder, or condition in an individual in need thereof throughadministering to the individual a therapeutically effective amount of anActRIIB polypeptide as described above. These methods are particularlyaimed at therapeutic and prophylactic treatments of animals, and moreparticularly, humans.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes prophylaxis of the named condition or amelioration orelimination of the condition once it has been established.

In certain embodiments, compositions (e.g., soluble ActRIIBpolypeptides) of the invention are used as part of a treatment for amuscular dystrophy. The term “muscular dystrophy” refers to a group ofdegenerative muscle diseases characterized by gradual weakening anddeterioration of skeletal muscles and sometimes the heart andrespiratory muscles. Muscular dystrophies are genetic disorderscharacterized by progressive muscle wasting and weakness that begin withmicroscopic changes in the muscle. As muscles degenerate over time, theperson's muscle strength declines. In particular, muscular dystrophiescaused by loss of dystrophin function that can be treated with a regimenincluding the subject ActRIIB polypeptides include Duchenne musculardystrophy and Becker muscular dystrophy.

Duchenne muscular dystrophy (DMD) was first described by the Frenchneurologist Guillaume Benjamin Amand Duchenne in the 1860s and is one ofthe most frequent inherited diseases in males, affecting one in 3,500boys. DMD is caused by mutations or deletions in the dystrophin genethat prevent formation of full-length dystrophin protein. Males areparticularly at risk for dystrophin defects because they possess only asingle copy of the dystrophin gene, which is located on the Xchromosome. Dystrophin normally functions as a critical component of amultiprotein complex that connects the plasma membrane (sarcolemma) ofthe muscle cell (fiber) with the actin cytoskeleton and extracellularmatrix, thereby stabilizing the sarcolemma during fiber contractions. Inthe absence of functional dystrophin, the sarcolemma and eventually theentire muscle fiber are readily damaged during cycles of contraction andrelaxation. Early in the course of DMD, muscle compensates byregeneration, but eventually muscle progenitor cells cannot keep up withthe ongoing damage, and healthy muscle is replaced by non-functionalfibro-fatty tissue.

Becker muscular dystrophy (BMD) is named after the German doctor PeterEmil Becker, who first described this variant of DMD in the 1950s. BMDresults from different mutations in the dystrophin gene. BMD patientshave some dystrophin, but it is either insufficient in quantity or poorin quality. Having some dystrophin functionality protects the muscles ofthose with BMD from degenerating as badly or as quickly as those ofpeople with DMD.

Utrophin, an autosomal protein structurally similar to dystrophin, isbroadly expressed with dystrophin in the sarcolemma during embryonicdevelopment. Utrophin expression in muscle normally declines by the timeof birth and becomes restricted in mature fibers to neuromuscularjunctions and muscle-tendon junctions. Compelling evidence suggests thatutrophin could substitute for dystrophin and provide therapeuticbenefits in muscular dystrophy patients if a method can be devised toincrease utrophin levels along the sarcolemma, as in early development(Miura et al., 2006, Trends Mol Med 12:122-129). In an experimentalproof of principle, transgenic expression of utrophin in muscle resultedin complete recovery of normal mechanical function and prevention ofmuscular dystrophy in an mdx mouse model (Tinsley et al., 1998, Nat Med4:1441-1444).

Recent researches demonstrate that blocking or eliminating function ofGDF8 (an ActRIIB ligand) in vivo can effectively treat at least certainsymptoms in DMD and BMD patients. Thus, the subject ActRIIB polypeptidesmay act as GDF8 inhibitors (antagonists), and constitute an alternativemeans of blocking the functions of GDF8 and/or ActRIIB in vivo in DMDand BMD patients. This approach is confirmed and supported by the datashown herein, whereby an ActRIIB-Fc protein was shown to induce broadsarcolemmal expression of utrophin as it increases muscle mass andstrength in a mouse model of muscular dystrophy.

7. Pharmaceutical Compositions

In certain embodiments, compounds (e.g., ActRIIB polypeptides) of thepresent invention are formulated with a pharmaceutically acceptablecarrier. For example, an ActRIIB polypeptide can be administered aloneor as a component of a pharmaceutical formulation (therapeuticcomposition). The subject compounds may be formulated for administrationin any convenient way for use in human or veterinary medicine.

In certain embodiments, the therapeutic method of the invention includesadministering the composition topically, systemically, or locally as animplant or device. When administered, the therapeutic composition foruse in this invention is, of course, in a pyrogen-free, physiologicallyacceptable form. Further, the composition may desirably be encapsulatedor injected in a viscous form for delivery to a target tissue site(e.g., bone, cartilage, muscle, fat or neurons), for example, a sitehaving a tissue damage. Topical administration may be suitable for woundhealing and tissue repair. Therapeutically useful agents other than theActRIIB polypeptides which may also optionally be included in thecomposition as described above, may alternatively or additionally, beadministered simultaneously or sequentially with the subject compounds(e.g., ActRIIB polypeptides) in the methods of the invention.

In certain embodiments, compositions of the present invention mayinclude a matrix capable of delivering one or more therapeutic compounds(e.g., ActRIIB polypeptides) to a target tissue site, providing astructure for the developing tissue and optimally capable of beingresorbed into the body. For example, the matrix may provide slow releaseof the ActRIIB polypeptides. Such matrices may be formed of materialspresently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalciumphosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, methods of the invention can be administered fororally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Certain compositions disclosed herein may be administered topically,either to skin or to mucosal membranes. The topical formulations mayfurther include one or more of the wide variety of agents known to beeffective as skin or stratum corneum penetration enhancers. Examples ofthese are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide,dimethylformamide, propylene glycol, methyl or isopropyl alcohol,dimethyl sulfoxide, and azone. Additional agents may further be includedto make the formulation cosmetically acceptable. Examples of these arefats, waxes, oils, dyes, fragrances, preservatives, stabilizers, andsurface active agents. Keratolytic agents such as those known in the artmay also be included. Examples are salicylic acid and sulfur.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants which may be required. Theointments, pastes, creams and gels may contain, in addition to a subjectcompound of the invention (e.g., an ActRIIB polypeptide), excipients,such as animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a subject compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

In certain embodiments, pharmaceutical compositions suitable forparenteral administration may comprise one or more ActRIIB polypeptidesin combination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions of the invention include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the subject compounds of the invention (e.g., ActRIIB polypeptides).The various factors will depend upon the disease to be treated. In thecase of muscle disorders, factors may include, but are not limited to,amount of muscle mass desired to be formed, the muscles most affected bydisease, the condition of the deteriorated muscle, the patient's age,sex, and diet, time of administration, and other clinical factors. Theaddition of other known growth factors to the final composition, mayalso affect the dosage. Progress can be monitored by periodic assessmentof muscle growth and/or repair, for example, by strength testing, MRIassessment of muscle size and analysis of muscle biopsies.

In certain embodiments of the invention, one or more ActRIIBpolypeptides can be administered, together (simultaneously) or atdifferent times (sequentially or overlapping). In addition, ActRIIBpolypeptides can be administered with another type of therapeuticagents, for example, a cartilage-inducing agent, a bone-inducing agent,a muscle-inducing agent, a fat-reducing, or a neuron-inducing agent. Thetwo types of compounds may be administered simultaneously or atdifferent times. It is expected that the ActRIIB polypeptides of theinvention may act in concert with or perhaps synergistically withanother therapeutic agent.

In a specific example, a variety of osteogenic, cartilage-inducing andbone-inducing factors have been described, particularly bisphosphonates.See e.g., European Patent Application Nos. 148,155 and 169,016. Forexample, other factors that can be combined with the subject ActRIIBpolypeptides include various growth factors such as epidermal growthfactor (EGF), platelet derived growth factor (PDGF), transforming growthfactors (TGF-α and TGF-β), and insulin-like growth factor (IGF).

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of ActRIIB polypeptides. Such therapy wouldachieve its therapeutic effect by introduction of the ActRIIBpolynucleotide sequences into cells or tissues having the disorders aslisted above. Delivery of ActRIIB polynucleotide sequences can beachieved using a recombinant expression vector such as a chimeric virusor a colloidal dispersion system. Preferred for therapeutic delivery ofActRIIB polynucleotide sequences is the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. Retroviral vectors can be madetarget-specific by attaching, for example, a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody. Thoseof skill in the art will recognize that specific polynucleotidesequences can be inserted into the retroviral genome or attached to aviral envelope to allow target specific delivery of the retroviralvector containing the ActRIIB polynucleotide. In one preferredembodiment, the vector is targeted to bone, cartilage, muscle or neuroncells/tissues.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for ActRIIB polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (see e.g., Fraley, etal., Trends Biochem. Sci., 6:77, 1981). Methods for efficient genetransfer using a liposome vehicle, are known in the art, see e.g.,Mannino, et al., Biotechniques, 6:682, 1988. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1. Generation of an ActRIIB-Fc Fusion Protein

Applicants constructed a soluble ActRIIb fusion protein that has theextracellular domain of human ActRIIb fused to a human or mouse Fcdomain with a minimal linker (three glycine amino acids) in between. Theconstructs are referred to as ActRIIb(20-134)-hFc andActRIIb(20-134)-mFc, respectively.

ActRIIb(20-134)-hFc is shown below as purified from CHO cell lines (SEQID NO: 5)

GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

The ActRIIb(20-134)-hFc and ActRIIb(20-134)-mFc proteins were expressedin CHO cell lines. Three different leader sequences were considered:

(i) Honey bee mellitin (HBML): (SEQ ID NO: 7) MKFLVNVALVFMVVYISYIYA(ii) Tissue Plasminogen Activator (TPA): (SEQ ID NO: 8)MDAMKRGLCCVLLLCGAVFVSP (iii) Native: (SEQ ID NO: 9)MGAAAKLAFAVFLISCSSGA.

The selected form employs the TPA leader and has the followingunprocessed amino acid sequence (SEQ ID NO: 29):

MDAMKRGLCCVLLLCGAVFVSPGASGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

This polypeptide is encoded by the following nucleic acid sequence (SEQID NO:10):

A TGGATGCAAT GAAGAGAGGG CTCTGCTGTG TGCTGCTGCTGTGTGGAGCA GTCTTCGTTT CGCCCGGCGC CTCTGGGCGTGGGGAGGCTG AGACACGGGA GTGCATCTAC TACAACGCCAACTGGGAGCT GGAGCGCACC AACCAGAGCG GCCTGGAGCGCTGCGAAGGC GAGCAGGACA AGCGGCTGCA CTGCTACGCCTCCTGGCGCA ACAGCTCTGG CACCATCGAG CTCGTGAAGAAGGGCTGCTG GCTAGATGAC TTCAACTGCT ACGATAGGCAGGAGTGTGTG GCCACTGAGG AGAACCCCCA GGTGTACTTCTGCTGCTGTG AAGGCAACTT CTGCAACGAG CGCTTCACTCATTTGCCAGA GGCTGGGGGC CCGGAAGTCA CGTACGAGCCACCCCCGACA GCCCCCACCG GTGGTGGAAC TCACACATGCCCACCGTGCC CAGCACCTGA ACTCCTGGGG GGACCGTCAGTCTTCCTCTT CCCCCCAAAA CCCAAGGACA CCCTCATGATCTCCCGGACC CCTGAGGTCA CATGCGTGGT GGTGGACGTGAGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGGACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGAGGAGCAGTAC AACAGCACGT ACCGTGTGGT CAGCGTCCTCACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACAAGTGCAAGGT CTCCAACAAA GCCCTCCCAG TCCCCATCGAGAAAACCATC TCCAAAGCCA AAGGGCAGCC CCGAGAACCACAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCAAGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTATCCCAGCGAC ATCGCCGTGG AGTGGGAGAG CAATGGGCAGCCGGAGAACA ACTACAAGAC CACGCCTCCC GTGCTGGACTCCGACGGCTC CTTCTTCCTC TATAGCAAGC TCACCGTGGACAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCCGTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT GTCTCCGGGT AAATGA

N-terminal sequencing of the CHO-cell produced material revealed a majorsequence of −GRGEAE (SEQ ID NO: 11). Notably, other constructs reportedin the literature begin with an −SGR . . . sequence.

Purification could be achieved by a series of column chromatographysteps, including, for example, three or more of the following, in anyorder: protein A chromatography, Q sepharose chromatography,phenylsepharose chromatography, size exclusion chromatography, andcation exchange chromatography. The purification could be completed withviral filtration and buffer exchange.

ActRIIb-Fc fusion proteins were also expressed in HEK293 cells and COScells. Although material from all cell lines and reasonable cultureconditions provided protein with muscle-building activity in vivo,variability in potency was observed perhaps relating to cell lineselection and/or culture conditions.

Example 2: Generation of ActRIIB-Fc Mutants

Applicants generated a series of mutations in the extracellular domainof ActRIIB and produced these mutant proteins as soluble fusion proteinsbetween extracellular ActRIIB and an Fc domain. The backgroundActRIIB-Fc fusion has the sequence (Fc portion underlined)(SEQ IDNO:12):

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Various mutations, including N- and C-terminal truncations, wereintroduced into the background ActRIIB-Fc protein. Based on the datapresented in Example 1, it is expected that these constructs, ifexpressed with a TPA leader, will lack the N-terminal serine. Mutationswere generated in ActRIIB extracellular domain by PCR mutagenesis. AfterPCR, fragments were purified through a Qiagen column, digested with SfoIand AgeI and gel purified. These fragments were ligated into expressionvector pAID4 (see WO2006/012627) such that upon ligation it createdfusion chimera with human IgG1. Upon transformation into E. coli DH5alpha, colonies were picked and DNAs were isolated. For murineconstructs (mFc), a murine IgG2a was substituted for the human IgG1. Allmutants were sequence verified.

All of the mutants were produced in HEK293T cells by transienttransfection. In summary, in a 500 ml spinner, HEK293T cells were set upat 6×10⁵ cells/ml in Freestyle (Invitrogen) media in 250 ml volume andgrown overnight. Next day, these cells were treated with DNA:PEI (1:1)complex at 0.5 ug/ml final DNA concentration. After 4 hrs, 250 ml mediawas added and cells were grown for 7 days. Conditioned media washarvested by spinning down the cells and concentrated.

Mutants were purified using a variety of techniques, including, forexample, protein A column and eluted with low pH (3.0) glycine buffer.After neutralization, these were dialyzed against PBS.

Mutants were also produced in CHO cells by similar methodology.

Example 3: Generation of Truncated Variant ActRIIB(25-131)-hFc

Applicants generated a truncated fusion protein, ActRIIB(25-131)-hFc(FIGS. 7-8), which exhibits effects on muscle that are similar to thoseobserved with ActRIIB(20-134)-hFc. ActRIIB(25-131)-hFc was generatedusing the same leader and methodology as described above with respect toActRIIB(20-134)-hFc. The mature ActRIIB(25-131)-hFc protein purifiedafter expression in CHO cells has the sequence shown below (SEQ ID NO:23):

ETRECIYYNA NWELERTNQS GLERCEGEQD KRLHCYASWRNSSGTIELVK KGCWLDDFNC YDRQECVATE ENPQVYFCCCEGNFCNERFT HLPEAGGPEV TYEPPPTGGG THTCPPCPAPELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQDWLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLPPSREEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK

Example 4: Effect of ActRIIB-Fc on Muscle Mass and Strength in Mdx Mice

In order to determine the ability of ActRIIB(20-134)-Fc protein toincrease muscle mass in a disease condition, applicants determined theability of the ActRIIB-Fc protein to increase muscle mass in the mdxmouse model of muscular dystrophy.

Adult mdx mice were treated twice/week with ActRIIB(20-134)-mFc protein(1, 3, or 10 mg/kg; intraperitoneal) or a PBS vehicle control. The forcea mouse exerts when pulling a force transducer is measured to determineforelimb grip strength. The average force of five pulling trials wasused for the comparison of grip strength between the cohorts. At the endof the study, femoris, gastrocnemius, pectoralis and diaphragm muscleswere dissected and weighed. Grip strength measurements showed asignificant increase also. The muscle mass results are summarized in thetable, below.

Tissue Weights from Vehicle- and ActRIIB(20-134)-mFc-Treated Mdx Mice

Gastrocnemius Femoris Pectoralis Dia- (L + R) (L + R) (L + R) phragmVehicle-treated Average (grams) ± 0.413 ± 0.296 ± 0.437 ± 0.111 ± Std.deviation 0.040 0.019 0.060 0.030 ActRIIB(20-134)-mFc (10 mg/kg) Average(grams) ± 0.52 ±  0.39 ± 0.807 ± 0.149 ± Std. deviation 0.050 0.05 0.210.020 Ttest p-value 0.0006 0.0006 0.002 0.05

As illustrated in the table, the ActRIIB(20-134)-mFc-treated groupsexhibited increased lean tissue mass in the mdx mice compared to thePBS-treated mice. ActRIIB-Fc treatment increased gastrocnemius size25.9%, femoris size 31.8%, and pectoralis muscles by 85.4% compared tothe vehicle control group. Of possible clinical importance, we alsofound that the diaphragm weights of ActRIIB(20-134)-mFc-treated micewere increased 34.2% compared to the control cohort. These datademonstrate the efficacy of ActRIIB-Fc protein in a muscular dystrophydisease condition.

Additionally, mdx mice treated with ActRIIB-Fc protein exhibit increasedgrip strength compared to the vehicle-treated controls. At 16-weeks, the1, 3 and 10 mg/kg ActRIIB-Fc groups demonstrated a 31.4%, 32.3% and64.4% increase in grip strength, respectively, compared to the vehiclecontrol group. The improved grip strength performance of theActRIIB(20-134)-mFc-treated groups supports the idea that the increasedmuscle found in the treatment groups is physiologically relevant. Mdxmice are susceptible to contractile—induced injury and undergosignificantly more cycles of degeneration and regeneration than theirwild-type counterparts. Despite these muscle phenotypes,ActRIIB(20-134)-mFc treatment increases grip strength in the mdx mice.

In Duchenne's Muscular Dystrophy, disease onset occurs early inchildhood, often as early as age five. Accordingly, the data presentedabove with respect to adult mice do not necessarily reflect the effectsan ActRIIB molecule would have in children with DMD. To address this, astudy was conducted with juvenile mdx mice.

ActRIIB(20-134)-mFc treatment significantly increases body weight injuvenile (four week old) C57BL/10 and mdx mice. Body compositionanalysis using in vivo NMR spectroscopy revealed increased lean tissuemass accompanied the higher body weights. C57BL/10 mice treated withActRIIB(20-134)-mFc gained 35.2% lean tissue mass and the treated mdxgroup gained 48.3% more lean tissue mass than their respective controlcohorts. Further, the effect of ActRIIB(20-134)-mFc treatment onstrength was assessed. Vehicle-treated mdx mice grip strength scoreswere 15.7% lower than the vehicle C57BL/10 cohort thereby illustratingthe muscle weakness associated with dystrophin deficiency. In contrast,mdx mice treated with ActRIIB(20-134)-mFc improved their grip strengthcompared to the mdx vehicle group, and attained grip strengthmeasurements which surpassed C57BL/10 vehicle mice and reached the levelof the treated C57BL/10 grip strength scores (vehicle mdx: 0.140±0.01KgF; treated mdx: 0.199±0.02 KgF; vehicle C57BL/10: 0.166±0.03;0.205±0.02 KgF). Remarkably, the treatment restored the juvenile mdxmice back to wild type levels of grip strength. Therefore, theActRIIB(20-134)-mFc molecule is likely to have important clinicalapplications in Duchenne muscular dystrophy, particularly in juvenilepatients at an age close to the onset of the disease.

Example 5: Effect of ActRIIB-Fc on Sarcolemmal Expression of Utrophin inMdx Mice

The most common types of muscular dystrophy are caused by partial orcomplete loss of functional dystrophin protein, leading to fragility ofthe sarcolemma (muscle cell membrane), muscle weakness, and eventualmuscle necrosis. Utrophin is a structurally similar protein, albeit witha highly restricted distribution in mature muscle fibers under normalconditions. Compelling evidence suggests that utrophin could substitutefor dystrophin and provide therapeutic benefits in many musculardystrophy patients if a method can be devised to increase utrophinlevels along the entire sarcolemma of muscle fibers, as is the caseduring early development (Miura et al., 2006, Trends Mol Med12:122-129).

Therefore, Applicants investigated the ability of ActRIIB(20-134)-mFc toincrease utrophin levels throughout the sarcolemma of muscle fibers inan mdx^(5cv) mouse model, in which a point mutation in exon 10 of thedystrophin gene creates a premature stop codon and dysfunctionaldystrophin protein. Beginning at 4-6 months of age, mdx^(5cv) mice weretreated with ActRIIB(20-134)-mFc, 10 mg/kg, s.c., or vehicle(Tris-buffered saline) twice per week for 20 weeks. Upon termination ofdosing, the pectoralis major and extensor digitorum longus (EDL) muscleswere removed and frozen for later analysis.

Effects of ActRIIB-Fc on utrophin expression in muscle were investigatedby Western blot analysis and immunohistochemistry. In preparation forthe former, pectoralis major muscles were homogenized mechanically witha hand-held tissue homogenizer in the presence of protease andphosphatase inhibitors. Protein samples were run on 4-12% acrylamideNuPAGE® Novex® Tris mini gel (Invitrogen) and transferred ontoImmobilon®-FL polyvinylidene fluoride membrane (Millipore). Equalprotein loading among lanes and uniform protein transfer acrossmembranes were confirmed with Ponceau stain prior to immunodetection.Membrane-bound utrophin was detected with a murine monoclonal antibodydirected against recombinant human utrophin (MANCHO3 clone 8A4, diluted1:200; Developmental Studies Hybridoma Bank, University of Iowa). Thisantibody has been shown to recognize mouse utrophin, as well as human,dog, and Xenopus homologs. The secondary antibody was a rabbitanti-mouse antibody conjugated to horseradish peroxidase (diluted1:2000). Densitometry was performed with a Chemi Genius BioimagingSystem (Syngene), and utrophin levels were normalized to GAPDH(glyceraldehyde 3-phosphate dehydrogenase) level in each sample tocontrol for nonuniform processing. For immunohistochemical analysis,utrophin was visualized on acetone-fixed transverse sections (14 μmthickness) of EDL muscle fibers with a murine primary antibody directedagainst the C-terminus of human utrophin (Santa Cruz Biotechnology,catalog no. sc-81556) and a goat anti-mouse secondary antibody labeledwith Alexa Fluor 488 (Invitrogen, catalog no. A21121).

These complementary approaches yielded strong evidence that ActRIIB-Fcinduces sarcolemmal expression of utrophin in mdx mice. As assessed byWestern blot, chronic treatment with ActRIIB(20-134)-mFc in middle-agedmdx mice increased utrophin protein levels by more than 80% compared tocontrols when averaged over the entire pectoralis muscle (FIG. 9).Moreover, immunohistochemical analysis of EDL muscle fibers confirmedthat the increase in utrophin expression was localized to thesarcolemma. As shown in FIGS. 10-11, utrophin was barely detectable inmost sarcolemmal segments of vehicle-treated mdx mice, as expected formature muscle fibers of mdx adults. Compared to controls, there was amarked increase in sarcolemmal utophin levels in age-matched mdx micetreated with ActRIIB(20-134)-mFc, with utrophin widely distributed alongthe sarcolemma of individual fibers (FIGS. 10-11). These resultsdemonstrate that ActRIIB(20-134)-mFc treatment can unexpectedly induce abroad sarcolemmal distribution of utrophin in muscle fibers in a mousemodel of muscular dystrophy. This capability to induce utrophin, andpotentially compensate for dystrophin deficiency, indicates thatActRIIB(20-134)-mFc treatment may ameliorate sarcolemmal instability andcontraction-related cellular damage as it increases muscle size andstrength. Therefore, in addition to providing patients with increasedmuscle mass and strength, treatment with antagonists of ActRIIBsignaling may create muscle fibers that are more resistant to damage anddegeneration that is typical of DMD and BMD.

Example 6: Effect of ActRIIB-Fc on Sarcolemmal Integrity and Force Dropwith Eccentric Contractions in Mdx Mice

Applicants will treat mdx^(5cv) mice with ActRIIB(20-134)-mFc or vehicleas described above to determine whether utrophin-inducing properties ofActRIIB-Fc protect against sarcolemmal instability andcontraction-related muscle damage in a mouse model of musculardystrophy. In addition, wildtype mice serving as controls will betreated with ActRIIB(20-134)-mFc or vehicle. A tracer assay with Evan'sblue dye will be used to assess use-dependent sarcolemmal permeability(as an indicator of membrane integrity) by detecting infiltration ofplasma serum albumin into muscle fibers. Mice in each group will beexercised regularly on a treadmill and then injected IP with sterilizedEvan's blue dye (50 μl of a 10 mg/ml buffered solution per 10 g bodyweight) at the end of dosing. Muscles will be excised twenty-four hourslater and immediately frozen in chilled isopentane. Transverse sectionswill be prepared with a cryostat and processed for microscopicvisualization of Evan's blue dye infiltration in individual fibers.Immunohistochemical staining for a sarcolemmal protein (such as laminin)may be used to confirm or reveal sarcolemmal boundaries, and thepercentage of total fibers that are infiltrated by serum albumin (Evan'sblue dye) may be quantified. Applicants expect that treatment withActRIIB(20-134)-mFc will ameliorate or prevent infiltration of Evan'sblue dye into muscle fibers of exercised mdx^(5cv) mice, as anindication of improved sarcolemmal integrity.

In the mdx mouse, skeletal muscles (particularly fast-twitch muscles)display an excessive drop in maximal tetanic force after a series ofeccentric contractions, during which muscle fibers lengthen as theyexert force (contract). This excessive drop in force has been attributedto contraction-dependent fiber injury stemming from disruption of adystrophin-deficient sarcolemma (Blaauw et al, 2008, Hum Mol Genet17:3686-3696). Therefore, additional mdx mice and wildtype controls willbe studied to determine whether ActRIIB-Fc can, through itsutrophin-inducing capability, provide resistance to fiber damagemediated by eccentric contractions. In this experiment, mdx^(5cv) mice(or wildtype controls) will be exercised on a treadmill at regularintervals and treated with ActRIIB(20-134)-mFc or vehicle as describedabove. At the cessation of dosing, muscles containing primarilyfast-twitch fibers (such as the EDL) will be excised and testedaccording to an ex vivo protocol similar to that of Krag et al, (2004,Proc Natl Acad Sci USA 101:13856-13860). In brief, muscles will beweighed and attached to both a micrometer and a force transducer withinan organ bath containing oxygenated Ringer's solution. Stimulation willbe performed with field electrodes connected to a stimulator unit. Theforce drop associated with eccentric contractions will be calculatedusing the difference of isometric force generation during the first andtwentieth tetanus of a standard protocol. At the end of physiologicaltesting, muscles will be quick frozen in chilled isopentane for laterhistological analysis. Applicants expect that treatment withActRIIB(20-134)-mFc will reduce the force drop associated with eccentriccontractions, as an indication of reduced sarcolemmal fragility.

Example 7: Effect of ActRIIB-Fc on Exercise Induced Muscle Damage in MdxMice

Applicants investigated the ability of ActRIIB(20-134)-mFc to blunt orreverse exercise induced muscle damage in an mdx^(5cv) mouse model. Fiveweek old mdx mice were divided into four groups (N=10 for each group).The first group was given no intervention, sacrificed after four weeksand assessed for serum creatine kinase levels. The second group wasgiven treadmill exercise and sacrificed after four weeks. The thirdgroup was given treadmill exercise for eight weeks, with vehicletreatment (TBS) given from week four through week eight. The fourthgroup was given treadmill exercise for eight weeks, withActRIIB(20-134)-mFc treatment (10 mg/kg, twice weekly) given from weekfour through eight.

Serum creatine kinase levels are shown for each group in FIG. 12.ActRIIB-Fc treatment completely prevented exercise induced damage (asmeasured by serum creatine kinase levels) accruing after week four, andmoreover, reversed the damage occurring prior to week four (comparegroup 2 versus group 4). Accordingly, ActRIIB-Fc prevents and reversesdamage to muscle fibers in a mouse model of Duchenne's musculardystrophy, consistent with the other findings herein with respect toincreased utrophin levels.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

1-52. (canceled)
 53. A method for treating a DMD or BMD patient, themethod comprising administering to the patient an effective amount of anantibody or antigen-binding fragment thereof that binds to ActRIIB, andwherein the patient has elevated levels of serum CK-MM relative to thenorm for patients with the same disease state.
 54. The method of claim53, wherein the patient has DMD
 55. The method of claim 53, wherein thepatient has BMD.
 56. The method of claim 53, wherein administration ofthe antibody or antigen-binding fragment thereof increases sarcolemmalstrength of muscle fibers in the patient.
 57. The method of claim 56,wherein utrophin expression is increased in the patient's skeletal orcardiac muscle.
 58. The method of claim 53, wherein the method furthercomprises evaluating a marker for muscle degeneration and selecting adose level or frequency based on the level of the marker for muscledegeneration, wherein the marker for muscle degeneration is serum CK-MM.59. The method of claim 53, wherein the method comprises administeringan effective amount of an antibody that binds to ActRIIB.
 60. The methodof claim 53, wherein the method comprises administering an effectiveamount of an antigen-binding fragment that binds to ActRIIB.
 61. Themethod of claim 53, wherein the antibody is humanized.
 62. The method ofclaim 53, wherein the antibody is a monoclonal antibody.